Nitric oxide (NO) is a pleiotropic signaling molecule that affects numerous biological functions. One unique posttranslational modification of proteins by NO is termed S-nitrosylation (SNO), but the role in modulating key proteins’ function in melanoma has not been fully characterized. In the present study, by conducting the biotin switch assay and mass spectrometry, we confirmed that p53 was S-nitrosylated under nitrosative stress. Further proteomic characterization showed that Cys242, Cys275, and Cys277 were S-nitrosylated in A375 cells treated with 100 µM GSNO.