Reduced glutathione (GSH, gamma-L-glutamyl-L-cysteinylglycine) is a fundamental low-molecular mass antioxidant that serves several biological functions. Upon enzymatic and non-enzymatic oxidation, GSH forms glutathione disulfide (GSSG) and, under particular conditions, may generate other oxidative products. The determination of GSH, its precursors, and metabolites in several bio-matrices is a useful tool in studying oxidative stress. Many separative and non-separative methods have been developed and improved for the assay of GSH and related compounds. At present, high-performance liquid chromatography and capillary electrophoresis are the most used separative techniques to determine GSH and congeners. The review will deal with analytical methods developed over the last few years for the determination of GSH and related compounds, and with the procedures performed in sample pre-treatment in order to minimize analytical errors. Since GSH, GSSG, and related compounds lack of strong chromophores or fluorophores, it is advantageous, in many assays, to derivatize the compounds in order to improve the detection limit with UV-Vis and to allow fluorescence, thus the most commonly used labeling agents are also described.