Determination of the ratio of reduced to oxidized glutathione is of profound clinical interest in assessing the oxidative status of tissues and body fluids. However, this ratio is not yet a routine clinical parameter due to the analytically challenging interconversion of reduced (free) glutathione to oxidized (bound) glutathione. We aimed to facilitate this ratio determination in order to aid its incorporation as a routine clinical parameter. To this end, we developed a simple derivatization route that yields different isotopologues of N-ethylmaleimide alkylated glutathione from reduced and oxidized glutathione (after its chemical reduction) for mass spectrometric analysis. A third isotopologue can be used as isotopic standard for simultaneous absolute quantification. As all isotopologues have similar chromatographic properties, matrix effects arising from different sample origins can only impact method sensitivity but not quantification accuracy. Robustness, simplified data analysis, cost effectiveness by one common standard, and highly improved mass spectrometric sensitivity by conversion of oxidized glutathione to an alkylated glutathione isotopologue are the main advantages of our approach. We present a method fully optimized for blood, plasma, serum, cell, and tissue samples. In addition, we propose production of N-ethylmaleimide customized blood collection tubes to even further facilitate the analysis in a clinical setting.