Abstract: American ginseng contains a variety of active ingredients. Bioactive peptides with biological functions can be obtained by protease hydrolysis, which has physiological activities such as lowering blood pressure, anti-oxidation and antibacterial. The inorganic salts, polysaccharides and free amino acids contained in the hydrolysate of protease will affect the biological activity of peptides to some extent, so it is necessary to further separate and purify the hydrolysate. At present, the commonly used peptides separation methods are time-consuming and labor-intensive, and the yield of target peptide components is low. In this study, large-scale separation and purification of proteins in a short time can be achieved by the protein purification system, which improves work efficiency and reduces costs. Biological activity and functional properties of peptides are stronger than those of protein. Therefore, it is of great significance to study the antioxidant peptides in freeze-dried American ginseng for nutritional evaluation and pharmacological effects. The amino acid composition of the peptides extracted from freeze-dried American ginseng was investigated by ultra-high performance liquid chromatography-quadrupole electrostatic field-Orbitrap tandem mass spectrometry (UHPLC-Q-Orbitrap MS/MS). The crude protein in freeze-dried American ginseng was extracted by alkali-soluble acid precipitation method, and hydrolyzed by various proteases. The optimal protease for enzymatic defrosting of the crude protein was screened out using DPPH free radical clearance as an evaluation index. The crude protein was enzymolized with the best protease, and different components were obtained by 3 ku dialysis bags. The components with better anti-oxidation activity were separated by protein liquid phase analysis system. The results showed that after pepsin hydrolysis for 1 h, DPPH free radical scavenging activity is the best, and the clearance rate is (56.02±2.01)%. Further, the antioxidant activity of <3 ku components is better than that of >3 ku components or endogenous peptides. The components with good antioxidant activity (<3 ku) were separated by ÄKTA^TMpure protein liquid phase analysis system to evaluate the DPPH free radical scavenging ability and hydroxyl free radical scavenging ability of each fraction, among which F1 and F2 have strong antioxidant activities. The amino acid composition was identified by UHPLC-Q-Orbitrap MS/MS. The hydrophobic amino acids such as Gly, Val and Leu are identified in F1 and F2, which may make them have good antioxidant activities. This method can quickly and effectively identify the antioxidant peptides composition in freeze-dried American ginseng, and provides a basis for revealing the relationship between antioxidant peptides composition and antioxidant activity.