A liquid chromatography-tandem mass spectrometric method（LC-MS/MS） was developed for the determination of content of vitamin A,vitamin D2，vitamin D3，vitamin E and vitamin K1 in infant formula milk.The fat vitamins were extracted with petroleum ether after enzymatic hydrolysis and saponification.The chromatographic separation was completed with the column of BEH C18（100 mm×2.1 mm,1.7 μm） using methanol（0.02% formic acid）-0.1% formic acid solution as mobile phase.Ionization mode was positive atmospheric pressure chemical ionization（APCI）.Mass spectrometry data was acquired by multiple reaction monitoring（MRM） mode.The quantification analysis was carried out by isotope dilution method（each vitamin had its isotope internal standard）.The calibration curves of 5 fat vitamins were linear in the certain concentration ranges with correlation coefficients（r2） above 0.99.The recoveries at three spiked levels（low,middle and high） ranged from 83.8% to 113.7% with relative standard deviations（RSD,n=6） of 1.2%-10.1%.The quantitation limits were in the range of 0.5-10 μg/100 g.The method was rapid,easy,accurate and sensitive,and could be used to detect fat vitamins in infant formula milk.