Abstract: The milk powder sample of 5 g was taken, dissolved by an appropriate amount of water at 40-45℃, and diluted to 100 mL by water. An aliquot (1 mL) was taken and mixed with 50 μL of 1 000 mg·L^-1 (calculated by choline-1,1,2,2-d₄) internal standard solution of choline chloride-1,1,2,2-d₄ and 10 mL of 1 mol·L^-1 hydrochloric acid solution, and the mixted solution was heated at 70℃ for 3 h. After cooling, the acidity of the above solution was adjusted to pH (5.0±0.1) by 500 g·L^-1 sodium hydroxide solution, and the solution obtained was diluted to 100 mL by water. An aliquot (1 mL) of the above solution was taken, diluted to 10 mL by mixed solution composed of acetonitrile and 10 mmol·L^-1 ammonium formate solution at volume ratio of 95:5, and passed through a 0.22 μm organic filter membrane. The filtrate was used for analysis by high performance liquid chromatograph-single quadrupole mass spectrometer. In chromatographic analysis, ACQUITY UPLC BEH HILIC column was used as the stationary phase, and mixed solutions composed of acetonitrile and 10 mmol·L^-1 ammonium formate solution-formic acid mixed solution (pH 5.0) at different volume ratios were used as the mobile phase for gradient elution. In mass spectrometry analysis, ESI⁺ mode was used for ionization, and SIM mode was used for detection. The internal standard method was used for quantification. It was shown by the results, linear relationship between values of the mass concentration and the peak area ratio of choline to choline-1,1,2,2-d₄ was kept in the range of 10.0-200 μg·L^-1, with detection limit (3S/N) of 1 mg/100 g. Test for recovery was made by standard addition method, giving recoveries in the range of 96.2%-100%. Tests for intra-day and inter-day precision were made on the 3 actual samples, and RSDs of determined values of choline were in the ranges of 0.90%-3.3% (n=11) and 1.6%-2.5% (n=9), respectively. The proposed method was applied to comparison with the enzyme colorimetric method in the national standard of GB 5413.20-2013, giving absolute values of relative errors between determined values of choline in the 15 actual milk powder samples not greater than 8.0%.