In this report, a pair of platinum wire electrodes was inserted in the main channel of a simple-cross PMMA microchip to perform on-chip alternating current-assisted proteolysis. Both terminals of the injection channel in the microchip were sharpened with a file to form sharp inlet tips to suck protein and trypsin solutions into the electrode-containing channel with the aid of a suction cup via the detection reservoir at the end of the chip. A solution containing BSA, cytochrome c, myoglobin, or hemoglobin was digested in the presence of alternating electric field after it was mixed with trypsin solution in the channel. The obtained digests were identified by MS with sequences that were comparable to those obtained by using conventional in-solution tryptic digestion while the digestion time was significantly reduced from 12 h to 4 min. The present proteolysis setup is simple and efficient and will find wide applications in high-throughput protein digestion.