Objective To explore the regulation of amino acid metabolism during rat liver regeneration (LR).    Methods Rats were randomly divided into 10 groups with 5 rats in each group, including nine partial hepatectomy (PH) groups and one normal control group. Selective reaction monitoring/multi reaction monitoring(SRM/MRM) was employed in the targeted metabolomics identification of 20 amino acids at 10 time points in rat liver regeneration. The change of amino acid content was analyzed by hierarchical clustering.    Results Alanine was up-regulated at 30, 36 and 72 hours; arginine was up-regulated at 6 and 12 hours; aspartic acid was up-regulated at 6, 12 and 36 hours; glutamate was up-regulated at 6, 12, 30, 36, 72 and 120 hours; histidine was up-regulated at 12, 24, 30, 36, 72 and 120 hours; glutamine was up-regulated at 72 hours, and isoleucine was down-regulated at 24 hours. Through hierarchical clustering analysis of amino acids，these amino acids can be grouped into three clusters.    Conclusion Many amino acids have changed during the liver regeneration, and throughout the whole process of rat liver regeneration. Changes in amino acids content play an important role in hepatocyte proliferation during the liver regeneration.