S-glutathionylation is an important post-translational modification of a cysteine residue of target protein. We applied a quantitative redox proteomics approach to identify the specific cysteine residues as targets of S-glutathionylation. Using this approach, we profiled CHAC1-associated changes in glutathionylome in APAP-induced hepatoxicity and ferroptosis. Primary mouse hepatocytes (PMHs) were infected with Ad-GFP or Ad-CHAC1 for 36 h, and then challenged with 20 mM APAP for 3 h followed by the enrichment and quantitative analysis of glutathionylated proteins.