Among the different 1,4-benzodiazepine urinary metabolites, those of bromazepam possess distinctive chemical features that may be used in their selective isolation and detection. The detection of bromazepam metabolites in urine was carried out using EMIT d.a.u., thin-layer chromatography (TLC), and gas chromatography-mass spectrometry (GC-MS). The positive EMIT d.a.u. benzodiazepine assay for bromazepam was found to be due to the 3-hydroxybromazepam (3HOB) metabolite. The detection by TLC and GC-MS was carried out after enzyme or acid hydrolysis of the glucuronide conjugates. Both the 2-amino-3-hydroxy5-bromobenzoylpyridine (AHBBP) metabolite and the acid hydrolysis product of 3-HOB, 2-amino-5-bromobenzoylpyridine (ABBP), were selectively detected by TLC. The bromazepam metabolites in urine could be both isolated and detected selectively by GC-MS in the presence of the metabolites of other 1,4-benzodiazepines that were sometimes used in combination with bromazepam. Both 3-HOB and AHBBP were detected by GC-MS only after trimethylsilyl (TMS) derivatization and not as the free compounds or the acetyl derivatives. Only ABBP was detected in three forms: ABBP, the TMS derivative, and the acetyl derivative. Evidence has been obtained from the enzyme hydrolysis and the TLC studies for the formation of the glucuronide conjugate of AHBBP at the 3-OH group rather than at the 2-NH2 group. All the results have been validated using reference 3-HOB and AHBBP.