Glutathione is a ubiquitous thiol-containing tripeptide, which plays a central role in cell biology. It is implicated in the cellular defence against xenobiotics and naturally occurring deleterious compounds, such as free radicals and hydroperoxides. Glutathione status is a highly sensitive indicator of cell functionality and viability. Its levels in human tissues normally range from 0.1 to 10 mM, being most concentrated in liver (up to 10 mM) and in the spleen, kidney, lens, erythrocytes and leukocytes. In humans, GSH depletion is linked to a number of disease states including cancer, neurodegenerative and cardiovascular diseases. The present review proposes an analysis of the current knowledge about the methodologies for measuring glutathione in human biological samples and their feasibility as routine methods in clinical chemistry. Furthermore, it elucidates the fundamental role of glutathione in pathophysiological conditions and its implication in redox and detoxification process. Several methods have been optimised in order to identify and quantify glutathione forms in human biological samples. They include spectrophotometric, fluorometric and bioluminometric assays, often applied to HPLC analysis. Recently, a liquid chromatography-mass spectrometry technique for glutathione determination has been developed that, however, suffers from the lack of total automation and the high cost of the equipment. Glutathione is a critical factor in protecting organisms against toxicity and disease. This review may turn useful for analysing the glutathione homeostasis, whose impairment represents an indicator of tissue oxidative status in human subjects.