Proteolysis is an important part of protein identification in proteomics analysis. The conventional method of in-solution digestion of proteins is time-consuming and has limited sensitivity. In this study, trypsin- or alpha-chymotrypsin-immobilized microreactors prepared by a microfluidics-based enzyme-immobilization technique were studied for rapid sample preparation in proteomic analysis. The kinetic studies for hydrolysis of substrate by microreactors revealed that immobilized proteases had higher hydrolytic efficiency than those performed by in-solution digestion. The performance of the microreactors was evaluated by digesting cytochrome c and BSA. Protein digestion was achieved within a short period of time (approximately 5 min) at 30 degrees C without any complicated reduction and alkylation procedures. The efficiency of digestion by trypsin-immobilized reactor was evaluated by analyzing the sequence coverage, which was 47 and 12% for cytochrome c and BSA, respectively. These values were higher than those performed by in-solution digestion. Besides, because of higher stability against high concentration of denaturant, the microreactors can be useful for immediate digestion of the denaturated protein. In the present study, we propose a protease-immobilized microreactor digestion method, which can utilize as a proteome technique for biological and clinical research.