Reducing the capacity of a victim to react against sexual assault, coupled with a possible abrupt unconsciousness-inducing effect and ease of administration in spiked drinks, have resulted in the use of sedative agents in cases of drug-facilitated offence. Among these compounds, tetrazepam may impair an individual rapidly. The chances of detecting this substance increase if the most sensitive methods are used and if the biologic matrix that allows the longest possible detection time is available. To document the window of detection of tetrazepam, 50 mg of the drug was administered orally to 2 volunteers, and the following samples were collected: oral fluid (n = 1) over 515 minutes, urine (n = 2) over 236-240 hours, hair (n = 2) 4 weeks after exposure, and beard (n = 1) over 34 days. Tetrazepam was analyzed by LC-MS/MS (Micromass Quattro Micro) after alkalinization and extraction by dichloromethane/diethyl ether in the presence of diazepam-d5, used as internal standard. Reversed-phase separation on an XTerra MS C18 column was achieved in 12 minutes, under gradient conditions. Pseudo-molecular ions selected were m/z 289.2 and 290.2 for tetrazepam and the internal standard (IS), respectively, and the corresponding daughter ions selected were m/z 225.2 and 253.2 for tetrazepam and m/z 154.1 and 198.3 for the IS. Urine tested positive for tetrazepam over 236-240 hours (14-13 ng/mL). Oral fluid tested positive for tetrazepam over 515 minutes (2.5 ng/mL). Tetrazepam was detected in beard over 27 days (6.5 pg/mg). A single tetrazepam dose was detected in hair 4 weeks after intake (123-175 pg/mg). Tetrazepam tested positive over the studied time intervals but would be expected to be detectable for a considerably longer time. Therefore, in cases of drug-facilitated crimes in which tetrazepam is involved, hair and beard analyses can be an important complement to urine analyses to document exposure, particularly if LC-MS/MS is used.