Glutathione, the very important intracellular antioxidant, is present in intracelullar environment in milimolar concentrations. Glutathione is a tripeptide molecule, which plays an essential role in the antioxidant system, as well as in maintenance of the intracellular redox state. This thiol compound exists in two forms, the reduced (GSH) and the oxidized (GSSG), and the ratio of both forms is crucial for the characterization of the oxidative stress in cells. Number of analytical methods have been developed for the measurement of the glutathione. Especially, High Performance Liquid Chromatography methods (HPLC) are mostly used linked to different types of detection, including electrochemical, UV/VIS or fluorimetric detection. Another approach for glutathione assay is using the spectral methods, either fluorimetric or spectrophotometric assays. In enzymatic assay, glutathione reductase reduces GSSG with simultaneous oxidation of specific substrate, which is sequentially photometrically detected. The fluorimetric method is based on the detection of derivatized GSH molecule.