A sensitive high-performance liquid chromatographic method with fluorescence detection for determining methamphetamine and its major metabolite, amphetamine, in abusers’ hair segments was developed. Methamphetamine and amphetamine in hair samples collected from addicts were extracted into acidified methanol, derivatized with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride, separated isocratically on an ODS column using TRIS–HCl buffer (0.1 mol dm^–3, pH 7.0)–methanol (30 + 70 v/v) as the mobile phase and the derivatives were detected fluorimetrically at 440 nm (λ_ex 330 nm). Calibration curves obtained by using control human hair spiked with standard solutions were linear (r ≥ 0.999) up to at least 676.1 ng mg^–1 for amphetamine and 746.1 ng mg^–1 for methamphetamine. The detection limits at a signal-to-noise ratio of 3 were 51.4 and 74.6 pg mg^–1 hair for amphetamine and methamphetamine, respectively. Using control hair spiked with standard solutions, the intra- and inter-day relative standard deviations (n = 5) were ≤8.6% for both the target compounds. The method was successfully applied to the segmental analyses of methamphetamine abusers’ hair samples.