TL;DR: DOT Drug Test Collection Process
49 CFR Part 40—standardized nationwide for all DOT testing
Urine (minimum 45 mL) or oral fluid per new DOT rules
90-100°F within 4 minutes of collection
Bottle A (primary) and Bottle B (split) required
15-20 minutes typical for standard collection
8415 Pineville-Matthews Rd, Suite 102, Charlotte, NC 28226
At Charlotte DOT Exam Center, our collectors have performed thousands of federal drug test collections since 1991. Dr. Alan Tebby and Dr. Lemuel Byrd ensure our collection staff follows every protocol required by 49 CFR Part 40—because errors in collection can invalidate results and create compliance problems for everyone involved. Our 84+ years of combined oversight experience and 4.8-star rating reflect our commitment to getting it right the first time.
The DOT drug test collection process is standardized across the entire country. Whether you’re tested in Charlotte or California, the same federal protocols apply. Understanding this process helps you know what to expect and ensures a smooth experience that gets you back to work without delays or complications.
Collection errors can void your test, delay results, or trigger “shy bladder” protocols that add hours to your day. Our collectors perform DOT specimen collections daily—not as an occasional task squeezed between other clinic duties. This experience means faster processing, fewer errors, and proper handling from start to finish.
Federal Collection Requirements: 49 CFR Part 40
49 CFR Part 40 establishes the procedures for all DOT-regulated drug testing collections. These rules apply regardless of which DOT agency regulates your employer—FMCSA, FAA, FTA, FRA, or PHMSA all follow identical collection protocols.
The regulations govern every aspect of collection: who can collect, where collection can occur, what forms must be used, how specimens must be handled, and what happens when problems arise. This standardization ensures consistency and legal defensibility across millions of annual DOT drug tests.
Step-by-Step Collection Process
Here’s exactly what happens during a standard DOT drug test collection:
Present valid photo ID (driver’s license, passport, or government-issued ID with photo and signature). The collector verifies your identity against the testing paperwork.
You’ll be asked to empty your pockets and leave personal items with the collector. Remove jackets, hats, and bags. This prevents introducing materials that could adulterate the specimen.
The collector prepares the Federal Drug Testing Custody and Control Form (CCF). You’ll verify your information is correct on the form before collection begins.
You select a sealed collection cup from available supplies, verifying the seal is intact. This ensures the container hasn’t been tampered with.
Enter the restroom with only the collection cup. Provide at least 45 mL of urine. Don’t flush until the collector instructs you. The water in toilets may be blued to prevent dilution.
Within 4 minutes of collection, the collector checks specimen temperature (90-100°F acceptable range). Out-of-range temperature requires immediate recollection under direct observation.
The collector splits your specimen into Bottle A (primary, 30 mL minimum) and Bottle B (split, 15 mL minimum) in your presence. Both bottles are sealed with tamper-evident tape.
Initial the bottle seals and sign the CCF. You receive Copy 5 for your records. The collector packages the specimen for shipment to the HHS-certified laboratory.
The Chain of Custody Form (CCF)
The Federal Drug Testing Custody and Control Form is the legal document that tracks your specimen from collection through final disposition. It’s a multi-part carbonless form:
CCF Copy Distribution
- Copy 1: Laboratory copy (travels with specimen)
- Copy 2: MRO copy (for Medical Review Officer review)
- Copy 3: Collection site copy (remains with collector)
- Copy 4: Employer copy
- Copy 5: Donor copy (you keep this)
Keep your copy. If questions arise about your collection, this documents exactly what happened and when.
Direct Observation Collection
Certain circumstances require the collector to directly observe you providing the specimen:
Direct observation must be performed by a same-gender observer. If a same-gender collector isn’t available, a same-gender observer is brought in specifically for this purpose.
Shy Bladder Procedures
If you cannot provide the minimum 45 mL specimen:
- Discard the insufficient specimen
- Begin a 3-hour waiting period
- Drink up to 40 oz of fluids during this time
- Attempt collection again
- If still insufficient, the MRO evaluates for medical explanation
- Without valid medical explanation, it’s treated as a refusal
To avoid shy bladder situations, stay moderately hydrated before your test—but don’t overhydrate, as excessively dilute specimens can also cause problems.
What Can Go Wrong During Collection
Understanding potential issues helps you avoid them:
Collection cannot proceed without proper identification. Expired IDs may not be accepted.
Triggers immediate observed recollection. Often indicates substitution attempt.
Treated as refusal to test with full consequences.
Leaving the collection site before completing all steps is a refusal.
Oral Fluid Testing: The New Option
In 2023, DOT finalized rules permitting oral fluid (saliva) testing as an alternative to urine testing. Key differences:
- Collection method: Saliva swab instead of urine specimen
- Observed collection: All oral fluid collections are observed (the collector watches you swab)
- Detection window: Shorter than urine—typically detects recent use within hours to days
- Same substances: Tests for the same 5-panel substances
- Employer choice: Employer decides whether to use urine or oral fluid testing
Note: As of early 2026, HHS-certified laboratories for oral fluid testing are still limited. Most DOT testing continues to use urine collection.
Frequently Asked Questions About DOT Drug Test Collection
How long does the DOT drug test collection process take?
A standard collection typically takes 15-20 minutes from check-in to completion. Factors that can extend this time include waiting for available collectors, shy bladder procedures (up to 3 hours), or direct observation requirements. Plan for extra time if your employer schedules multiple drivers simultaneously.
What should I bring to my DOT drug test?
Valid photo ID (driver’s license, passport, or government-issued ID with photo and signature). If your employer provided paperwork, bring that too. Leave unnecessary personal items in your vehicle—you’ll need to empty your pockets anyway.
Can I eat or drink before my DOT drug test?
Yes. Unlike some medical tests, you don’t need to fast. However, moderate your fluid intake—excessive hydration can result in dilute specimens that require recollection. Normal eating and drinking is fine.
What if I’m on my period during the collection?
Menstruation doesn’t prevent urine collection. Inform the collector, who may provide additional guidance. The laboratory accounts for potential contamination during the analysis process.
Can I request a specific collector gender?
For standard collections, any trained collector can perform the collection. For direct observation collections (return-to-duty, follow-up, or triggered by temperature/tampering issues), a same-gender observer is required—this isn’t optional for either party.
What happens to my specimen after collection?
The collector packages your sealed specimen and ships it via courier to an HHS-certified laboratory. The laboratory performs initial immunoassay screening. Negative results are reported. Presumptive positives undergo GC-MS confirmation testing. Confirmed positives go to the MRO for review before final reporting.
Schedule Your DOT Drug Test Collection
Charlotte DOT Exam Center provides compliant DOT drug test collections for all testing types. Our experienced collectors follow 49 CFR Part 40 protocols precisely, ensuring valid collections that won’t create downstream problems.
Walk-ins welcome Monday through Friday from 9 AM to 5 PM and Saturday from 9 AM to 12 PM. Employers: Contact us about account setup for streamlined driver testing.
Call 704-544-3494 or visit us at 8415 Pineville-Matthews Road, Suite 102, Charlotte, NC 28226.
For comprehensive information about all DOT drug testing requirements, visit our main DOT Drug Testing Charlotte NC hub page.
