Glutathione is a natural tripeptide that plays a major role in different physiological processes in the human body. Determination of glutathione in different body fluids and tissues is essential for early diagnosis and follow-up of various diseases. The assay of glutathione is problematic because of the high polarity, the limited stability, and the aliphatic structure, which lacks the appropriate chromophore for UV detection. A number of methods have been reported for determination of glutathione using different techniques. High-performance liquid chromatography was employed in both reverse phase and hydrophilic interaction modes. Electrochemical methods exploited the redox activity of glutathione to allow for quantification by different electrodes after chemical modification, including glassy carbon, carbon paste, and nanocomposite electrodes. Capillary zone electrophoresis was used with less need for derivatization which makes it simpler, faster, and more economic. A number of nanosensors and probes have been developed to assay glutathione in biological fluids using semiconductor nanoparticles, quantum dots, genetically engineered green fluorescent probes, and new derivatives of known dye classes. This work is an updated review of the methods of analysis of glutathione and glutathione disulfide in pharmaceuticals and biological fluids with more emphasis on the technical problems and the assay artifacts.