We have developed a GC-MS-MS assay for GHB in human hair. Five milligrams of washed hair were hydrolyzed by 1M or 0.01M NaOH before a liquid-liquid extraction with ethyl acetate under acidic conditions. GHB-d(6) was used as the internal standard. TMS derivatives were formed before injection. TBDMS derivatives were used in cases of strong chromatographic interferences or in a confirmatory procedure. Analysis of basal levels of GHB in 61 drug-free donors gave the following results: the mean measured concentration for blond hair was 0.60 ng/mg (n = 12), SD = 0.19 ng/mg, and extreme figures were in the range 0.35-0.95 ng/mg. For brown hair, the mean measured concentration was 0.90 ng/mg (n = 30), SD = 0.42 ng/mg, and extreme figures 0.41-1.86 ng/mg. For black hair, the mean measured concentration was 0.90 ng/mg (n = 19), SD = 0.37 ng/mg, and extreme figures 0.32-1.54 ng/mg, showing no significant differences depending on hair color. Analysis of basal levels of GHB of 12 or more specimens in segmented hair showed a mean concentration of 1.22 ng/mg (0.31-8.4 ng/mg) and a relative standard deviation for each individual ranging from 6.75% to 37.98%. GHB was administered to a healthy 53-year-old white male (light brown hair) at oral dosages of 30, 45, and 60 mg/kg. Beard hair was collected just before administration and 24 h after (and each day for one week for the last dose), and a 7.5-cm scalp hair lock was collected 7 days after the last dose. A rise in GHB concentration was observed in beard hair for the 45 and 60 mg/kg dosages with a maximum at 24 h, whereas no change was observed for the 30 mg/kg dosage. Scalp hair was segmented into 3-mm long segments. The three proximal last segments showed significantly (0.0005 < p < 0.005) different concentrations of GHB (1.22, 1.27, and 1.66 ng/mg, respectively) when compared with the basal physiological level of GHB in this same person (mean = 0.62 ng/mg, SD = 0.15 ng/mg). A case of daily GHB abuse during bodybuilding allowed us to determine a concentration of GHB of 14 ng/mg, in a 2-cm long segment (black hair). A case of rape under the influence of GHB was documented through hair analysis (black hair) and positive analysis of the glass she used. Sampled 7 days after the sexual assault, the three last 3-mm long proximal segments tested for GHB exhibited concentrations of 3.1-5.3 and 4.3 ng/mg, respectively, whereas the mean physiological level determined in this woman was 0.71 ng/mg, SD = 0.17 ng/mg. The authors advise a two-step hair sampling as evidence of GHB consumption: the first sample at the time of exposure to show the contamination by sweat of the proximal segment in case of recent administration with a significant rise of hair level at the root, and the second after at least 3 or 4 weeks to avoid this contamination and determine the levels incorporated in the hair matrix before, during, and after the exposure.