A method is described here for the determination of total glutathione (TGSH) and glutathione disulphide (GSSG) in the seminal plasma of the male partners of couples requesting a fertility evaluation. A suitable sample preparation procedure prior to high-performance liquid chromatography analysis is discussed. After adequate sample preparation, the samples were derivatised with ortho-phthaldialdehyde to form a stable, highly fluorescent tricyclic derivative. Reversed-phase column chromatography was used for the separation, and the effluent was monitored with a fluorescence detector at an excitation wavelength of 350 nm and an emission wavelength of 420 nm. The analytical performance of this method was satisfactory. The intra-assay and inter-assay coefficients of variation were below 10%. The recoveries were as follows: 94.1% (CV 2.3%) for TGSH and 93.2% (CV 4.0%) for GSSG. No significant differences were found in either TGSH or GSSG concentration between the smokers and nonsmokers (2.07 ± 1.28 μm versus 1.56 ± 1.20 μm, P = 0.431 and 95 ± 56 nm versus 112 ± 138 nm, P = 0.825).