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Glutathione is a tripeptide, present in plants and other organisms in free (GSH) and dimer (GSSG) forms, known for its antioxidant activity. A high-throughput, economical method was developed to measure the total glutathione (TG) concentration in grape juice, using an enzymatic assay (EA), based on the reaction of thiol with 5,5′-dithio-(2-nitrobenzoic acid) (DTNB) in the presence of glutathione reductase enzyme. This method was automatised to allow high-throughput measurements in the concentration range of 1–100 mg L−1. GSH and GSSG were also quantified separately using the UHPLC-MS/MS method. The two methods (EA and UHPLC-MS/MS) gave comparable results in grape juice (R2 =0.97), where the concentrations of SO2 were low (<100 mg L−1). The sample preparation is a critical step in the quantitative analysis of glutathione given the high reactivity of GSH. In this work, ascorbic acid and SO2, commonly used in oenology, were tested as protecting agents at different concentration. The results given by the EA method could be altered by the reaction of SO2 and DTNB, however this effect was observed only at high concentration of SO2 (1 g L−1). Ascorbic acid at a 2.5 mg L−1 concentration protected the sample well, without interfering with the analysis, and allowed for storage for up to four months at − 20 °C.