This study was designed to investigate the effect of cooking on in vitro digestibility and peptide profiling of pork protein. We simulated gastrointestinal digestion of cooked pork that was treated with pepsin alone or followed by trypsin treatment. Digested products were identified using matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry and liquid chromatography–mass spectrometry analyses. Cooking led to a reduction (p < 0.05) in digestibility and band intensities on sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels. Peptide profiling and identification analyses also showed significant difference (p < 0.05) in the m/z ranges and number of peptides from the pepsin-digested products between raw (4 °C) and very well done samples (100 °C). Peptides sequenced from pepsin-digested samples under lower degrees of doneness disappeared as the temperature increased. Meanwhile, the trypsin cleavages appeared more consistent among different degrees of cooking. Further work may be needed to evaluate the bioavailability of the digested products under different cooking temperatures.