Abstract: In order to establish a reliable method for quantitative analysis of vitamin D in infant milk powder, a ultra performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) with isotopes dilution method was developed. The samples were saponifed with KOH and extracted by pentane-ethyl ether solution (V(pentane)∶V(ethyl ether)=4∶1). The target analytes were separated on the Waters T₃ column with gradient elution using methanol and water (containing 2 mmol/L ammonium formate for both) as mobile phase. The identification and quantification were achieved by using electrospray ionization in positive ion mode and with multiple reaction monitoring. Internal standard method was used to determine the results. The linear range of the calibration curve was 5—150 μg/L. The limit of detection (LOD) of the method was 1.5 μg/kg, and the limit of quantitation (LOQ) was 5.0 μg/kg. The recoveries of the method were in the range of 97.0%—104.7% and 92.7%—108.0% for vitamin D₂ and vitamin D₃ respectively. The relative standard deviations (RSD) of intra-day and inter-day were less than 10%. The simply, rapid, accurate and sensitive method can be applied to determine the vitamin D in infant milk powder.