Migraine is a headache condition found in significant frequency in the general population. One recent study has shown that riboflavin, Vitamin B2, is an effective prophylactic treatment for this headache condition. One subject in a recent study conducted by the Division of Forensic Toxicology, Armed Forces Institute of Pathology (AFIP) was taking 200 mg of riboflavin twice daily for the prevention of migraine headaches. When that subject's urine was tested using Abbott TDx drugs-of-abuse assays a number of tests resulted in a MX BKG error and all samples had BLK I values greater than those observed with normal urine specimens. The MX BKG error occurs when the BLK I value is greater than the upper limit determined by the manufacturer for a particular assay. High BLK I values may result if the specimen being analyzed contains a fluorophore that will compete with the fluorescein-labeled antibody used in the assay. This error serves as a notification that an interfering substance may be present and the assay is not performing according to manufacturer-specifications. Upon termination of riboflavin therapy the subject's BLK I values began to decrease within 60 h of the last 200 mg dose. A second subject began chronic riboflavin use to confirm this interferent effect. Elevated BLK I values resulted within 3 h of a single 200 mg dose and MX BKG errors occurred 1 h after a second 400 mg dose. No false negative results were noted with either subject (both subjects used butalbital and the first subject also used hydrocodone and diazepam during the study), suggesting that riboflavin is not an adulterant. Riboflavin use, however, does interfere with the TDx DAU assays and may result in quantitative values being determined which are of questionable validity in the face of an elevated BLK I value or may result in only an MX BKG error and no quantitative value reported. It is unclear if the interfering fluorophore is simply riboflavin itself or a combination of riboflavin and its metabolic products. Results obtained on urine samples collected from individuals using prophylactic riboflavin for migraine prevention and analyzed by TDx may be of questionable validity. Such samples may require analysis utilizing another immunoassay technique that does not employ a fluorescein-labeled antibody.