Objective:To establish an analytical method for the simultaneous determination of five fat-soluble vitamins in serum using isotope dilution ultra high performance liquid chromatography-tandem mass spectrometry (ID-UPLC-MSMS).Methods:Fat-soluble vitamins were obtained from serum samples which collected from Shanghai Tenth People′s Hospital between April 2019 and August 2019 by the extraction method, and were detected by ID-UPLC-MSMS. The performance of the method was verified by referring to the relevant documents of the Clinical and Laboratory Standards Institute (CLSI).Results:The ID-UPLC-MSMS method for the rapid detection of various fat-soluble vitamins in serum was proposed and successfully verified. The linear range of the method: vitamin A: 25-2 500 μg/L, 25(OH)D 2: 2-200 μg/L, 25(OH)D 3: 2-200 μg/L, vitamin E: 0.25-50 mg/L, vitamin K1: 0.1-20 μg/L. The intra- and inter-assay precision standard deviations of the five analytes were within ± 15%, and the accuracy of the test results of the 25(OH)D 2 and 25(OH)D 3 standards was 96.44%-102.37%. Conclusion:The performance of ID-UPLC-MSMS method for the simultaneous determination of five fat-soluble vitamins is satisying, and the result is accurate and reliable, which suggested it can be used for the clinical sample.