The generation of ratiometric, glutathione specific, redox-sensitive variants of fluorescent probes enable real-time monitoring of intracellular redox changes by making it possible to cancel out most or all of the possible variability caused by instrument efficiency and effective dye content. The probes permit an indirect measure of the glutathione intracellular redox potential EG, achieved by using the Nernst equation after the measurement of intracellular glutathione redox state. However, EG is strongly dependent on pH and on the total concentration of glutathione, and since their intracellular spatial distribution is not isotropic, the measurement of EG may be affected by systematic errors. Here, we show that these errors are not negligible and we suggest possible methods to cancel them out when measuring the redox state of the glutathione redox couple