Disulfide bridge peptide mapping is used to determine the number of disulfide bonds and free sulfhydryl groups as well as the position and ratio of disulfide bond linkage in the biological pharmaceutical products. In this analysis, non-reduced samples are detected for intact disulfide bridges and reduced samples (for example, DTT treatment) are usually detected the disulfide bridges converted to free cysteine thiol groups. Both non-reduced and reduced samples are digested with 2-4 proteolytic enzymes. The digested peptides are then analyzed with the reverse-phase analytical HPLC system. Disulfide bond-linked peptides are determined by the ESI-MS/MS with powerful software based on the specific features of the disulfide bridges, as well as the difference in the chromatogram between non-reduced and reduced samples. The ratio of disulfide bond linked peptides and free cysteine residues also can be calculated according to the HPLC-MS/MS analysis.