In this study, the hydrolytic amino acids of Chinese human and Holstein cow milk of different lactation periods were analyzed by the advanced isobaric tags for relative and absolute quantitation (iTRAQ) combined with the high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) approach. The total contents of the hydrolytic amino acids in cow colostrum, cow mature milk, human colostrum and human mature milk were 5.00, 4.38, 2.12 and 2.48 g ? L-1, respectively. Among these, the contents of the hydrolytic amino acids in cow milk were higher than those in human milk, indicating a decreasing trend with the prolongation of lactation. Additionally, principal component and hierarchical cluster analyses were used to further screen the differentially expressed amino acids. These results enhanced the understanding of the hydrolytic amino acids in cow and human milk across different lactation periods, which could provide potential directions for newborn dairy powder and nutritious supplementary.