Abstract: Objective To develop a method for measurement of trimethylamine-N-oxide (TMAO)-microbiota-generated metabolite of choline in serum by stable isotope dilution high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and to study its accuracy and stability. Methods Proteins in samples were precipitated with three volumes of 10 μmol/L internal standard d9-TMAO in acetonitrile and supernatants were analyzed after injection onto a SiO2 column (2.1 mm×100 mm, 5 μm.) and equilibrated with 30% solution A (10 mmol/L ammonium formate, pH = 3.0) and 70% solution B (acetonitrile). Results Lower limit of detection was 0.003 μmol/L and the lower limit of quantitation was 0.063 μmol/L at linear range of 0.16 - 20.00 mol/L (r2=0.999 9). The intra-batch and inter-batch coefficients of variance were 2.03% - 2.64% and 6.00% - 9.94%, and the recovery rates were 89.5% - 103.0%, respectively. Stability study revealed that the storage temperature and time could affect the concentration of TMAO. TMAO in serum was stable both during storage at -80 ℃ and throughout multiple freeze thaw cycles. Conclusion HPLC-MS/MS assay has characteristics of rapidness, accuracy, precision, and high-throughput nature and can meet the detection requirements for large quantity of blood samples.