Cysteine modifications have been widely detected among different protein in different species. Here, we examined protein glutathionylation in HeLa cells under both untreated and diamide-treated conditions. A strategy using resin-assisted enrichment of glutathionylated proteins or peptides after biotin-switch is commonly used for detection of glutathionylation and other reversible cysteine modifications, and was also applied in this study. Using these strategies we identified a large number of glutathionylated protein with modification sites under both untreated and diamide-treated conditions.