Although non‐thermal discharge plasma is more and more applied in biological field, the molecular mechanisms of plasma acting on biomolecules are still unclear, which are indispensable for understanding the plasma‐induced biological effects. In this work, discharge plasma at the gas–solution interface was employed to irradiate on reduced glutathione (GSH), the most abundant low molecular weight thiol‐containing antioxidant in cells. Through Raman spectroscopy, both the irreversible damage and reversible conversion of GSH to oxidized glutathione (GSSG) were monitored in a non‐destructive fashion, so that the reaction kinetic processes through multi‐pathways could be evaluated quickly and quantitatively. Based on the experimental data, the reaction mechanism was discussed. magnified image