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Summary: Glutathione (GSH) is a highly dynamic, high abundance molecule regulating redox homeostasis in most mammalian cells. Traditional methods could not achieve quantification of glutathione in live cells with high spatial and temporal resolution. Here, we provide protocols on how to use reversible reaction-based ratiometric fluorescent probes, RealThiol (RT) and its derivatives, to quantify GSH globally or in specific organelles. The protocols are applicable to cultured or harvested cells through confocal imaging and flow cytometry.For complete details on the use and execution of this protocol, please refer to Chen et al. (2017) and Jiang et al. (2015, 2017, 2018a).