The overall study of post-translational modifications (PTMs) of proteins is gaining strong interest. Beside phosphorylation and glycosylation, truncations of the nascent polypeptide chain at the N- or C-terminus are by far the most common types of PTMs. Nevertheless, little attention has been paid to the development of approaches that allow a systematic analysis of these proteolytic processing events. Here we present a protocol that allows the identification of the C-terminal sequence of proteins separated by two-dimensional polyacrylamide gel electrophoresis (2DE). For each purified protein, a peptide mixture is generated by cleavage of the protein with cyanogen bromide. During incubation with carboxypeptidases only the original C-terminal fragment forms a ladder. Ladder readout is performed using MALDI mass spectrometry. 2DE-separated proteins from Shewanella oneidensis were chosen as a model system to investigate the effectiveness of the approach.