A method for the determination of testosterone in human hair by gas chromatography-mass spectrometry using d3-testosterone as internal standard is described. Our method consisted of alkaline digestion, fast liquid–liquid extraction, LH-20 chromatography and derivatization with heptafluorobutyric anhydride. Quantification was achieved by selected ion monitoring of m/z 680 (testosterone) and m/z 683 (d3-testosterone). Our method needed no complex corrections for isotope contributions. The procedure provided a sensitive and specific technique with good accuracy and precision. For the first time, testosterone has been quantified by gas chromatography-mass spectrometry in human hair. The concentrations (median, range, ng g–1 hair) reflected a significant (p = 0.05; t-test) sex difference with 2.7 ng g–1 (2.5–4.2) in male and 1.7 ng g–1 (1.0–3.4) in female hair.