In this report, infrared (IR)-assisted on-plate proteolysis has been developed for rapid peptide mapping. Protein solutions containing trypsin were allowed to digest directly on the spots of matrix-assisted laser desorption/ionization (MALDI) plates under IR radiation. The feasibility and performance of the novel proteolysis approach were investigated by the digestion of bovine serum albumin (BSA) and cytochrome c (Cyt-c). It was demonstrated that IR radiation substantially enhanced the efficiency of proteolysis and the digestion time was significantly reduced to 5 min. The digests were identified by MALDI time-of-flight mass spectrometry with sequence coverages of 55 (BSA) and 75% (Cyt-c) that were comparable to those obtained by using conventional in-solution tryptic digestion. The suitability of IR-assisted on-plate proteolysis to complex proteins was demonstrated by digesting human serum and casein extracted from commercially available milk sample. The present proteolysis strategy is simple and efficient, offering great promise for high-throughput protein identification.