Abstract: UHPLC-MS/MS was applied to the determination of N-terminal peptide of recombinant bovine somatotropin in milk. The sample solution was diluted to constant volume with the mixture of acetonitrile and 0.05% (φ) formic acid solution (4+6). Waters ACQUITY BEH-C18 column was used as stationary phase, and the mixture of acetonitrile and 0.05% (φ) formic acid solution mixed in different ratio was used as mobile phase in gradient elution. ESI+ and multi-reactions monitor were adopted in MS/MS. Linear relationship between values of peak area and mass concentration of N-terminal peptide of recombinant bovine somatotropin was obtained in the range of 0.1-2 mg·L-1, with lower limits of determination(10S/N) of 0.02 mg·kg-1. On the base of blank milk sample, test for recovery was made by standard addition method;values of recovery found were in the range of 85.0%-88.6%, with RSD′s (n=6) less than 9%.