Glutathione participates in maintaining the redox balance by cycling between the reduced (GSH) and oxidized (GSSG) forms. The ratio of concentrations of reduced and oxidized forms of glutathione is used as a cellular and tissue marker of oxidative stress. The aim of this work was to optimize the method for quantification of glutathione. In the paper, the chemical and biological approach to measuring glutathione in extracted samples of the heads of fruit flies were examined. The colorimetric method of measuring glutathione uses Ellman's reagent to determine the concentrations of reduced, oxidized and total glutathione in a sample. The validity of the method was tested by the influence of chemical compounds with the prooxidant, methamphetamine (METH), and the antioxidant, quercetin (QUE). Using a biological approach, we measured the shift in fluorescence between the reduced and oxidized forms of roGFP in response to exogenous reductants (DTT) and oxidants (DA). The results of the colorimetric method did not show statistically significant differences. There is a trend of increasing GSH/GSSG in the group treated with METH and its decrease in the group treated with QUE. Measurements using the biological approach of GSH/GSSG did not show differences between the untreated group and the groups treated with oxidant and reductant. The optimization of the method for measuring the reduced and oxidized form of glutathione was partially successful. Considering the expected trends obtained by the colorimetric method, we believe that further optimization will lead to reliable and statistically significant results. The use of the roGFP indicator is not optimal for measuring redox status in tissue homogenates, but primarily for in situ measurements for which it was primarily developed.