Objective To develop a method based on ultra-high-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) for the simultaneous determination of nine penicillin antibiotics residues in Nujin pill. Methods The samples were separated on the CQUITY UPLC BEH-C18(2.1 mm × 50 mm, 1.7 μm) chromatographic column by gradient elution using 0.1% formic acid andacetonitrle as the mobile phase.The electrospray ion source was operated in the positive ion mode using the multi-reaction monitoring(MRM) methed, and the results were quantified by the external standard method. Results The correlation coefficients(r) of the nine drugs were all greater than 0.999 in the corresponding mass concentrationranges, and the detection limit was 0.01～0.03 ng·mL-1 and the quantitative limit was 0.02～0.1 ng·mL-1.The average recovery(n=6) was 75.4%～100.2% and RSD was 0.54%～3.43%. Of the 30 samples tested, 1 was found to contain dicloxacillin at a level of 0.193 mg·kg-1. No other drugs were found. Conclusion The method is accurate, simple, sensitive and highly effective. It can be used for the determination of nine kinds of penicillin residues in traditional Chinese medicine pills.