Protein S-nitrosylation is the covalent redox-related modification of cysteine sulfhydryl groups with nitric oxide, creating a regulatory impact similar to phosphorylation. Recent studies have reported a growing number of proteins to be S-nitrosylated in vivo resulting in altered functions. These studies support S-nitrosylation as a critical regulatory mechanism, fine-tuning protein activities within diverse cellular processes and biochemical pathways. In addition, S-nitrosylation appears to have key roles in the etiology of a broad range of human diseases. In this review, we discuss recent advances in proteomic approaches for the enrichment, identification, and quantitation of cysteine S-nitrosylated proteins and peptides. These advances have provided analytical tools with the power to interpret the impact of S-nitrosylation at the system level, providing a new platform for drug discovery and the identification of diagnostic markers for human diseases.