Abstract: We utilized a multi-step derivatization gas chromatography-mass spectrometry method for the determination of common amino acid enantiomers, combined with deuterated hydrochloric acid hydrolysis, to identify nine trace D-amino acids in thymalfasin. We optimized the conditions for multi-step derivatization, the volume of reagent for redissolving samples, and the conditions for chromatography and mass spectrometry with isopropanol and trifluoroacetic anhydride as derivatization reagents, and validated the procedure, including sensitivity, linear range, precision, accuracy and recovery. Sixteen pairs of D/L-amino acids and glycine derivatives were separated within 29 min, with the limit of quantification as low as 0.09-2.79 μmol·L^-1. Nine amino acid derivatives of thymalfasin showed a good linear relationship within the concentration range examined (r²>0.992 3). The precision results showed that RSD values were less than 10.90%. Accuracy test results of a reference substance ranged from 76.69% to 128.18%. Average recoveries of spiked samples ranged from 70.41% to 125.39%. For the nine D-amino acids assayed, D-Asp and D-Glu content in six batches of thymalfasin were highest, ranging 0.41%-0.49% and 0.25%-0.33%, respectively, with others less than 0.25%. The method is sensitive, efficient and reliable, available for seventeen common amino acids and their enantiomers, and works well with simultaneous determination of nine trace D-amino acids in thymalfasin, providing a reference for the comprehensive control of racemic peptide impurities in this synthetic polypeptide drug.