The disulfide bond is one of the most common post-translational modifications in proteins, of which determination is essential to the comprehensive understanding of protein structures. Disulfide bond analysis has undergone great improvement due to the development of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), especially in terms of speed and sensitivity. In general, the characterization of disulfide-containing peptides is achieved by the reduction of disulfide bonds followed by alkylation. In this review we focus on the analysis of disulfide-containing proteins/peptides by some unique methods in MALDI MS. The MALDI in-source decay (ISD) of disulfide bonds and adducts of the matrix and sulfhydryl-containing peptide are discussed in detail. The mechanism of each method is discussed so as to help the reader gain greater insight into it, and examples of its application are also presented. The goal of this review is to provide an understanding of these techniques for analysis of disulfide-linked proteins/peptides in MALDI MS.