A method which uses the sequential reduction of glutathione using the enzyme glutathione reductase, and oxidation using DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) to produce the yellow product TNB (5-mercapto-2-nitrobenzoic acid) that can be quantitated using a spectrophotometer. The process of recycling the glutathione through multiple rounds of reduction and oxidation increases the sensitivity of the assay. The assay can be used to measure total reduced and oxidized glutathione (GSH + GSSG) or, by initially masking the endogenous GSH, the differential levels of the two components.