In this study, Q Exactive quadrupole Orbitrap mass spectrometry with Proteome Discoverer 2.2 software was used to identify the signature peptides of heat load from α-lactalbumin and β-lactoglobulin in heat-treated cow milk, and the effect of heating temperature on their contents was studied. The results showed that there was a correlation between the degree of whey protein denaturation and the contents of the signature peptides. With increasing heating temperature and time, the contents of the signature peptides increased significantly, and then remained unchanged when the heating temperature exceeded the denaturation temperature of whey protein. After heating at temperatures exceeding 80 ℃, the content of peptide 3# increased obviously, and it was found that the contents of peptides 1#-6# were significantly different between pasteurized milk and ultra high temperature (UHT) milk (P < 0.01). Therefore, based on ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and principal component analysis (PCA), this study can provide technical support for quality control and evaluation of thermally treated milk.