Objective To establish a method to quantify serum fat-soluble vitamins by liquid chromatography-tandem mass spectrome-try and evaluate their performance in preliminary clinical application.Methods The contents of fat-soluble vitamin in serum were quantified by liquid chromatography-tandem mass spectrometry.The samples were collected from 1 113 pregnant women from November 2022 to November 2023 at the Obstetrics Clinic of the First Affiliated Hospital of Soochow University.The method of liquid chromatog-raphy-tandem mass spectrometry for determination of fat-soluble vitamins in serum was validated referring to"Consensus of method de-velopment and validation of liquid chromatography-tandem mass spectrometry in clinical laboratories".Results The linear ranges of vitamin A,E,D2,D3 and K1 in serum were from 40 to 4 000 ng/mL,0.5 to 50 μg/mL,2 to 200 ng/mL,5 to 250 ng/mL and 0.1 to 10 ng/mL,respectively.The detectable limit was 2.50 ng/mL,0.10 ng/mL,0.40 ng/mL,1.00 ng/mL and 0.02 ng/mL,respec-tively.The limit of quantitation was 10.00 ng/mL,0.50 ng/mL,1.00 ng/mL,5.00 ng/mL and 0.10 ng/mL,respectively.The intra-batch coefficient of variation(CV)and inter-batch CV were all less than 15％.The rate of recovery was 91.25％to 107.18％,90.00％to 105.51％,92.88％to 107.87％,93.36％to 107.40％and 90.20％to 104.40％,respectively.The various fat-soluble vitamins in ser-um remained stable within 7 days under-20 ℃.The levels of fat-soluble vitamins in serum of pregnant women were detected by liquid chromatography-tandem mass spectrometry.There were significant differences of the levels and distributions for various fat-soluble vita-mins in the pregnant women in different age groups(P＜0.05),and the levels of fat-soluble vitamins gradually increased with the age.Conclusion The basic performance of the liquid chromatography-tandem mass spectrometry verified in this experiment was in line with the evaluation criteria,thus it should be highly sensitive and accurate for analyzing the contents of various fat-soluble vitamins in serum.