Only sporadic data are available on hair concentrations of diazepam and some of its metabolites (nordazepam, oxazepam, and temazepam) following a single controlled dose. The aim of this study was to investigate the deposition of diazepam and its metabolites in human hair after eight healthy volunteers (four women and four men, ages 24-26, East Asian) consumed 10 mg of diazepam. Hair was collected from all volunteers 1 month after exposure, and also 2 months post-exposure from men and 10 months post-exposure from women. Diazepam and the complete metabolite profile, including oxazepam glucuronide and temazepam glucuronide, were measured by ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with limits of quantifications (LOQs) of 0.5-2.5 pg/mg for diazepam, nordazepam, oxazepam, and temazepam, and of 10 pg/mg for oxazepam glucuronide and temazepam glucuronide. There were no differences by gender in the amounts of diazepam or metabolites found. The concentration of the main metabolite nordazepam was consistently higher than that of diazepam at both 1 and 2 months after consumption. Oxazepam and temazepam traces were found in some volunteers' hair, but the glucuronides were not detected. Diazepam and nordazepam levels at 10 months post-exposure were extremely low (near the LOQ), indicating drug loss by personal hygiene and physical handling. To our knowledge, this is the first single-dose diazepam study using black hair and the first study to include measurements of oxazepam glucuronide and temazepam glucuronide in human hair.