We developed an optimized sample collection protocol (patented) to minimize GSH artificial oxidation and a method which greatly improves sensitivity for GSSG, dramatically reducing starting material requirement. In our two-step alkylation approach, all GSH is initially blocked with N-ethylmaleimide (NEM), after which all small thiols are reduced (including GSSG). Newly released GSH is then re-alkylated with deuterated NEM (d5-NEM). A third isotopologue can be used as isotopic standard for absolute quantification. With all isotopologues having similar chromatographic properties, matrix effects can only impact method sensitivity but not quantification accuracy.