An ultra performance liquid chromatography-mass spectrometric（UPLC-MS）method was developed for the simultaneous determination of purine nucleotides（e.g.ATP，ADP，AMP，NAD+ and NADP+） in mammalian cell.The HPLC separation was carried out on a KinetexTM HILIC column（50 mm×4.6 mm,2.1 μm）.The experimental parameters,including-extraction method,compositions of mobile phase and MS parameters were optimized.The results demonstrated that the standard curves were linear in the range of 1.0-100 μmol/L,with correlation coefficients above 0.99.The average recoveries were in the range of 95.7%-101.1%with RSDs of 1.2%-6.1%.The method is sensitive and repeatable,and could be applied in the analysis of intracellular energy metabolism.