High-throughput proteomic analysis based on a biotin switch combined with liquid chromatography/tandem mass spectrometry (LC/MS/MS) enables simultaneous identification of S-nitrosylated sites and their cognate proteins in complex biological mixtures, which is a great help in elucidating the functions and mechanisms of this redox-based post-translational modification. However, detergents such as sodium dodecyl sulfate (SDS) and Triton X-100 adopted in these systems, which are hard to fully remove in the subsequent MS-based analyses, can suppress the peptide signals and influence the SNO-Cys site identification and the reproducibility of the experiments. Here we developed a detergent-free biotin-switch method, which applied urea to replace detergents, and successfully combined it with LC/MS/MS in the analysis of S-nitrosylated proteins. With this approach, 44 SNO-Cys sites were specified on 35 distinct proteins in S-nitrosoglutathione (GSNO)-treated HeLa cell extracts of proteins with good reproducibility. The LC/MS performance was greatly improved as analyzed with Pep3D and the amount of samples for analysis reduced from 40 mg used in the literature to 3-5 mg. For S-nitrosylated targets detected both in the control sample and in the GSNO-treated sample, extracted ion chromatography (XIC) was employed to estimate the quantitative change of S-nitrosylation (S-nitrosation), which facilitates the judgment on 'accept or reject' of the identified targets.