Abstract: Ultra performance liquid chromatography- tandem quadrupole mass spectrometry ( UPLC- TQMS) method had been developed for the determination for pantothenic acid (VB5) , biotin (VB7) , cyanocobalamine (VB12) in formula milk powder. The samples were ultrasonic extracted by water, and precipitated protein by chloroform for analyzing by UPLC-MSMS. The analytes were separated by HSS T3 column. The mobile phase consisted of 10mmol/L ammonium acetate with 0.1% formic acid and acetonitrile. The samples were identified and quantified by multiple reaction monitoring (MRM) via positive electrospray ionization (ESI+) . The results showed that the limit of quantification of VB5 were 5μg/100 g, VB7were 2μg/100 g, VB12were 0.2μg/100 g. The recoveries at three spiked levels and the relative standard deviations were 79.4%⁸7.8% and 3.3%⁵.3%, respectively. This method was simple, time saving, sensitive and accurate in the determination of VB5, VB7, VB12 in formula milk powder.