Abstract: Disulfide bond in protein is an important post-translational modification and it is extremely essential for stabilizing the three-dimensional structure of proteins, maintaining the correct folded conformation and regulating their biological activities. Therefore, disulfide bonds assignment is of importance to understand life process and to develop protein pharmaceutical. Mass spectrometry technology can serve not only as a means cleaving the disulfide bonds, but also as an analysis tool assigning disulfide bonds connectivity. As a fragmentation means, mass spectrometry has various of MS/MS fragmentation modes. As a detector, it has the advantages of high sensitivity and high accuracy for mass spectrometry information. With these merits, mass spectrometry could fulfill the desire in the characterization of disulfide bonds. This paper summarized the mass spectrometric methods for locating disulfide bonds in proteins or peptides, according to the medium where the disulfide bonds were broken and the cleavage mechanism. Two cleavage mediums-(solution and gas phase) were divided. According to the cleavage mechanism, reduction/oxidation reaction using chemical reagents, electrochemical reduction based on electrode reaction, and post-column reduction were classified, in which disulfide bonds were cleaved in solution and the mass spectrometer was mainly used as an analysis tool. Meanwhile, molecular collision fragmentation including CID and HCD, electrons capture fragmentation (ECD and ETD), photons absorption fragmentation (e.g. MALDI-ISD and UVPD), and cleavage based on ion-ion reactions were summarized, in which disulfide bonds were cleaved in gas phase. For these methods, mass spectrometer was used not only as an analysis tool but also more importantly as a cleavage approach. This review included most disulfide bonds assignment methods and would facilitate bioinformatics-related researchers to choose an appropriate mass spectrometric method. Additionally, at the end of this paper, several new challenges in the field of disulfide bond assignment were put forward such as the characterization of multiple disulfide bonds in a short peptide, insufficient study of the disulfide bond artifacts, and the new methods based on novel instruments like UVPD. Moreover, in terms of methodological study, it would be promoted by the development of the new ionization and fragmentation technique producing new kinds of characteristic ions and corresponding data processing tools emerged in the future. Through this review, it was expected to develop more effective mass spectrometric methods for disulfide bonds characterization.