NO-mediated S-nitrosation of cysteine residues has been recognized as a fundamental post-translational modification. S-Nitrosation of endothelial cell (EC) proteins can alter function and affect vascular homeostasis. Trace amounts of S-nitrosoproteins in endothelial cells (ECs) in vivo coupled with lability of the S-nitroso bond have hindered a comprehensive characterization. We demonstrate a convenient and reliable method, requiring minimal sample, for the screening and identification of S-nitrosoproteins. ECs treated with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) were subjected to the biotin switch method of labeling, then detected by analytical Western blot-based two-dimensional gel electrophoresis (2-DE). More than 89 SNAP-increased S-nitrosoproteins were detected and 28 of these were successfully excised from preparative 2-DE gel and identified by LC-MS/MS. Moreover, the nitrosocysteine residue for each protein (HSPA9/368, beta-actin/16, TMP3/170, vimentin/328) was also determined, and the relative ratio of S-nitrosation/non-S-nitrosation for Cys328 of vimentin was estimated using MASIC software. By the combination of the biotin switch method with 2-DE and Western blot analysis, S-nitrosoproteins can be screened and characterized by MS, providing a basis for further study of the physiological significance of each S-nitrosoproteins.