The GSH/GSSG-Glo Assay is a luminescence-based system for the detection and quantification of total glutathione (GSH +GSSG), GSSG and GSH/GSSG ratios in cultured cells. A change in GSH levels is important in the assessment of toxicological responses and is an indicator of oxidative stress, potentially leading to apoptosis or cell death. The assay provides a simple, rapid multiwell-plate format where stable luminescent signals are correlated with either the GSH or the GSSG concentration of a sample directly in culture wells. Both total glutathione and GSSG determinations are based on the reaction where GSH-dependent conversion of a GSH probe, Luciferin-NT, to luciferin by a glutathione-S-transferase enzyme is coupled to a firefly luciferase reaction. Light from luciferase is dependent on the amount of luciferin formed, which is in turn dependent on the amount of GSH present. This makes the luminescent signal proportional to the amount of GSH. Determination of total glutathione and GSSG are performed in parallel reactions. In one configuration the assay reagents measure total glutathione using a reducing agent that converts all the glutathione, GSH and GSSG in a cell lysate to the reduced form, GSH. In a second configuration the assay reagents are used to measure only the oxidized form, GSSG. In this case, a reagent is added that blocks all the GSH while leaving the GSSG intact. This blocking step is followed by a reducing step that converts the GSSG to GSH for quantification in the luminescent reaction. Because the assays are performed directly on cells in culture wells, loss of GSH or GSSG is minimized, reducing variability.