Proteolysis is the key step for proteomic studies integrated with MS analysis. Compared with the conventional method of in-solution digestion, proteolysis by a protease-immobilized microreactor has a number of advantages for proteomic analysis; i.e., rapid and efficient digestion, elimination of a purification step of the digests prior to MS, and high stability against a chemical or thermal denaturant. This chapter describes the preparation of the protease-immobilized microreactors and proteolysis performance of these microreactors. Immobilization of proteases by the formation of a polymeric membrane consisting solely of protease-proteins on the inner wall of the microchannel is performed. This was realized either by a cross-linking reaction in a laminar flow between lysine residues sufficiently present on the protein surfaces themselves or in the case of acidic proteins by mixing them with poly-lysine prior to the crosslink-reaction. The present procedure is simple and widely useful not only for proteases but also for several other enzymes.