This study applied peptidomics to investigate potential biomarkers for evaluating pork-meat freshness. Meat samples stored at -2, 4, 10, and 25 °C were collected at specific time points to evaluate meat freshness indicators (color, total viable count, pH, and total volatile basic nitrogen). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profile was analyzed, and substantial protein degradation (myosin heavy chain, paramyosin, troponin) was detected at the end of storage, regardless of the temperature. Peptidomics analysis was performed using a UHPLC-LTQ-Orbitrap mass spectrometer, and the potential peptide marker MVHMASKE was filtered via multivariate analysis and quantified by parallel reaction monitoring combined with external standard quantitation. In addition, the relationship between peptide content and change in meat freshness was verified using real-life samples and the content of MVHMASKE showed an obvious decline during storage, presenting a period of pork meat from fresh to spoilage. This study provides favorable evidences to evaluate pork meat freshness by mass spectrometry-based pep-tidomics.