A new approach to sample preparation and enzymatic digestion in bottom-up proteomics has been developed using alginate-based hydrogel entrapment of enzymes. This hydrogel facilitates rapid and room-temperature digestions with multienzyme capabilities. Three methodologies were tested: within microcentrifuge tubes, <i>in situ</i> pipette tips, and automated robotic liquid handling. Factorial experimental design identified a 1 h, room temperature, pepsin-trypsin dual-enzyme digestion as optimal for sequence coverage …