In this report, alternating current-assisted on-plate proteolysis has been developed for rapid peptide mapping. Protein solutions containing trypsin were allowed to digest directly on the spots of a stainless steel MALDI plate with the assistance of low-voltage alternating current electricity. Alternating current (AC) was allowed to pass through the protein solutions via the MALDI plate and a platinum disc electrode. The feasibility and performance of the novel proteolysis approach were investigated by the digestion of BSA and cytochrome c (Cyt-c). It was demonstrated that AC substantially enhanced the efficiency of proteolysis and the digestion time was significantly reduced to 5 min. The digests were identified by MALDI-TOF MS with sequence coverages of 42% (BSA) and 77% (Cyt-c) that were comparable to those obtained by using conventional in-solution tryptic digestion. The present proteolysis strategy is simple and efficient, offering great promise for MALDI-TOF MS peptide mapping.