Characterization of disulfide structures is used to determine folding of recombinant proteins derived from prokaryotic cells and also to obtain information on the tertiary structure of native proteins derived from eukaryotic cells, e.g., mammalian cells. The former characterization tells us whether refolded proteins retain correct tertiary structure, while the latter information tells us how the native proteins are folded, e.g., domain structure, folding motif and homology of folded structure. Information on which cysteinyl groups are involved in disulfide bonds or present in the free form can lead to potential analogs that have increased stability against oxidation and disulfide exchange. Therefore, a reliable, sensitive method of disulfide structure determination must be established. In our laboratory disulfide linkages of several growth factors and receptors have been determined by various techniques including partial reduction, partial oxidation, proteolytic fragmentation, detection of diPTH-Cys, and matrix-assisted laser desorption mass spectrometry. The disulfide structure thus determined chemically are consistent with those from X-ray or NMR study.