Kinetics of in-gel digestion of proteins by modified and native trypsins was studied by MALDI TOF mass spectrometry using 18O-labeled peptides as internal standards. The effect of the temperature, enzyme concentration, digestion time, and surface area of gel pieces on the yield of digestion products was characterized. Based on the kinetic data, we developed a protocol that enabled the identification of gel-separated proteins with 30-min digestion time without compromising the peptide yield and the sensitivity compared to conventional protocols that typically rely upon overnight enzymatic cleavage. The accelerated digestion protocol was tested in identification of more than 120 proteins from budding and fission yeasts at the subpicomole level.