To establish the content determination method of amino acids and characteristic peptides in Galli Gigerii Endothelium Corneum(GGEC) and evaluate the quality of GGEC from different origins.Methods:High-performance liquid chromatography(HPLC) was used to measure the content of 12 amino acids in GGEC，while ultra-high-performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS) was employed to determine the levels of two characteristic peptides.Partial least squares discriminant analysis(PLS-DA) was used to analyze the results.Results:The relative standard deviation(RSD) for the total content of the 12 amino acids in 18 batches of GGEC ranged from 2.84% to 14.01%.The RSD for the levels of the two characteristic peptides，Chicken-derived peptide I and Chicken-derived peptide Ⅱ，were 47.13% and 14.62%，respectively，indicating some fluctuations.PLS-DA results showed that three batches of samples from Shanxi and three batches of samples from Liaoning were distinctly grouped into separate categories，while nine batches of samples from Shandong and three batches of samples from Zhejiang were grouped together.Conclusion:The established method for determining amino acids and characteristic peptides in GGEC effectively evaluate the quality of GGEC from different origins and can provide a reference for the quality assessment of GGEC and other animal-based medicinal materials.