The hypnotic benzodiazepine flunitrazepam (Rohypnol®) has been identified as the drug of choice for the purposes of “drugging” unsuspecting victims and raping them while they are under the influence of this substance. The objective of this paper was to study elimination of flunitrazepam and 7-aminoflunitrazepam in urine collected from ten healthy volunteers who received a single 2 mg oral dose of Rohypnol®, to determine how long after drug administration 7-aminoflunitrazepam can be detected. A highly sensitive NCI-GC-MS method for the simultaneous quantitation of flunitrazepam (LOQ 100 pg/mL) and 7-aminoflunitrazepam (LOQ 10 pg/mL) in urine was developed. All samples were screened for benzodiazepines using optimized micro-plate enzyme immunoassay. The highest concentrations of 7-aminoflunitrazepam (70–518 ng/mL) and flunitrazepam (0.7–2.8 ng/mL) in urine were observed 6 h after drug administration in nine subjects and after 24 h in one subject. In six subjects 7-aminoflunitrazepam was detected up to 14 days after flunitrazepam administration, in one subject up to 21 days and in three subjects up to 28 days. In urine samples collected from six volunteers, flunitrazepam was detected three days after Rohypnol® intake, in three subjects 24 h, and in one subject 5 days later. Benzodiazepine micro-plate enzyme immunoassay kit allowed the detection of flunitrazepam and metabolities 5 to 21 days after drug administration.