High-performance liquid chromatography has been used for measurement of the concentrations of total cysteine and cysteinylglycine in human urine. The method involved conversion of disulfides to their reduced counterparts by use of tris(2-carboxyethyl)phosphine hydrochloride, derivatization with 1-benzyl-2-chloropyridinium bromide, and ion-pairing reversed-phase high-performance liquid chromatographic separation with ultraviolet detection at 315 nm. The linearity of the method was validated in the ranges 50–300 and 5–50 µmol L−1 urine for cysteine and cysteinylglycine, respectively; regression coefficients were better than 0.999. The detection and quantitation limits were 0.2 and 0.5 µmol L−1, respectively, for both analytes. Intra-assay and inter-assay imprecision were below 6.0%, and accuracy was 98.95–100.80%. The method was applied to urine samples donated by apparently healthy volunteers.