Glutathione is an endogenous antioxidant known for its protective role in various oxidative stress-mediated diseases. It is abundantly present in all human cell types and its levels, reduced (GSH) and oxidized (GSSG) are frequently used to determine the oxidative state of biological systems (Marrocco et al., 2017). It is the ratio of these biomarkers that provide more comprehensive oxidative stress information about the tissues, as opposed to other kits measuring only one. While GSH/GSSG assay kits are available from several manufacturers, Promega’s GSH/GSSG-Glo™ Assay kit seems to be highly sensitive and can be used with a low number of cells. However, the Promega assay is designed to measure total glutathione (tGSH) and GSSG in cell lysate samples from different wells or separate 3-dimensional (3D) tissues (reconstructed human tissues grown at the air-liquid interface (ALI)) or precision-cut tissue slices, with different lysis reagents to be used for assessments of each molecule. Using this assay for 3D tissues is not cost-effective as researchers would need to double the number of tissues for the experiment just for this single oxidative stress ratio endpoint. In this protocol, we describe a simple lysis method for 3D tissues that enables the use of a single tissue for measuring both GSH and GSSG using the GSH/GSSG-Glo™ Assay. The 3D tissues can be lysed using sulfosalicylic acid and then the lysate supernatants can be used immediately or stored for use at a later time. Before running the Promega assay, the samples must be neutralized using HEPES buffer and diluted as necessary to get the signal within a linear range. The samples can then be used for measuring the tGSH and GSSG using Promega’s assay procedure. The entire procedure can be completed within 5 hours.