Based on the monolithic silica gel materials with hierarchical pore structure and on the SPE devices (MonoTip) developed thereof, a trypsin-immobilized monolithic silica in a pipette tip (MonoTip Trypsin) suitable for digesting proteins has been newly developed. The surface of monolithic silica fixed into the tip was chemically modified with trypsin via an aminopropyl group. Trypsin-immobilized monolith successfully performed a rapid digestion of reduced and alkylated proteins with only a few times pipetting operation for the pre-treatment procedure of chromatographic analysis. The novel solid-phase digestion tool using monolithic silica allows a high-throughput trypsin proteolysis of bio-substances in proteomics.