A simple method for the separation of disulfide-bonded polypeptides using two-dimensional diagonal gel electrophoresis is described. The first dimension involves standard SDS-PAGE of nonreduced proteins. The separated proteins are then subjected to reduction before electrophoresis in the second dimension. This technique has several applications. It can be utilized to detect the presence of disulfide-bonded polypeptides within a protein, as an assay to detect the presence of a protein known to contain disulfide-bonded polypeptides in a mixture of proteins, and as a means of separating disulfide-bonded polypeptides from proteins of similar molecular weight.