Objective: An ultra performance liquid chromatography-tandem mass spectrometry method was established for the simultaneous determination of multiple water-soluble vitamins in infant milk powder by isotope dilution coupled with ultrasonic extraction. Methods: Sample was dissolved in warm water, then extracted by ultrasonic and adjusted to pH 4.6 with hydrochloric acid and sodium hydroxide. After high-speed centrifugation, finally detected by Ultra Performance Liquid Chromatography-tandem Mass Spectrometry (UPLC-MS/MS). The ionization was carried out in the positive ion mode of the Electrospray Ionization Source (ESI), and the target fragment ions were collected in the Multiple Reaction Monitoring (MRM) mode. Results: The internal standard curve of 10 water-soluble vitamins exhibited a good linearity in the concentration range of 2.0~500.0 μg/L with the correlation coefficient r² were greater than 0.998 7. The recovery rates of the standard recovery experiment were in the range of 85.7%~102.0%, while the Relative Standard Deviation (RSD) ranged from 1.11% to 5.69%. The Limit of Detection (LOD) and Quantification (LOQ) of the method were 10~50 μg/kg and 30~150 μg/kg, respectively. Conclusion: The method was simple to operate, short time consumed, good recovery rate, high sensitivity and stable results that could be applied to the determination of water-soluble vitamin content in infant milk powder.