A controlled clinical study was performed to test how long after administration of a single dose of Rohypnol flunitrazepam and its major metabolite could be detected in the urine and hair of volunteers, using previously developed NCI-GC-MS confirmatory technique and a commercially available micro-plate EIA. The micro-plate immunoassay method detected flunitrazepam and related compounds in urine at least up to 5 days after administration of a single dose of Rohypnol. This significant increase in the detection time interval is possible if the enzymatic hydrolysis of urine and solid-phase extraction are applied. The micro-plate enzyme immunoassay is recommended as a preliminary screening technique for urine in toxicological investigations of drug-facilitated sexual assault when the use of benzodiazepines is suspected. The application of solid-phase extraction and highly sensitive gas chromatography-mass spectrometry with negative ion monitoring chemical ionization detected 7-aminoflunitrazepam, flunitrazepam major metabolite, in urine 14 days after administration of a single dose of Rohypnol. The maximum concentration of this metabolite in urine was observed 6 to 24 hours after administration of a single dose of Rohypnol. The concentrations of 7-aminoflunitrazepam in hair were much higher than concentrations of the parent drug, flunitrazepam. The metabolite remained in hair for at least 1 month after administration of a single dose of Rohypnol. The study concluded that the 7-aminoclonazepam was being deposited in hair in much higher quantities than the parent drug and remained there for an extended period. The new technology detected the other compounds such as diazepam, alprazolam, triazolam, and ketamine in hair in very low concentrations. 14 tables