A high-performance liquid chromatographic method is presented for determination of glutathione in whole blood. Sample preparation involves hemolysis, protein precipitation, centrifugation, and filtration. The glutathione in the filtrate is then separated from other sulfhydryl-containing molecules by liquid chromatography with Zipax SCX cation-exchanger followed by detection with a mercury-based electrochemical detector. The liquid-chromatographic analysis time is approximately 5 min. Because of the chromatographic separation and the selectivity of the detector, the detection step is free from interferences from other components of blood. The method has been checked by comparison with the colorimetric assay based on reaction with 5,5'-dithiobis(2-nitrobenzoic acid). The chromatographic results are consistently slightly lower, presumably because of the greater selectivity of this method.