Abstract: The study aims to establish a quantitative analysis method for detecting betaine in red sugarbeet. By using the modified QuEChERS method combined with liquid chromatography-tandem mass spectrometry, the sample was extracted with methanol through accelerated solvent extraction(ASE), and purified by solid phase extraction, detected by hydrophilic interaction liquid chromatography- tandem mass spectrometry under multiple reaction monitoring mode and quantified by the external standard method. The results showed that linearity range was 0.05-5 μg/mL in the test. When the adding level was 4-16 mg/g, the recovery was 87.7%- 95.7%, and RSD was between 3.6% and 8.4%. The limit of detection (LOD) and the limit of quantification (LOQ) were 0.01 and 0.04 mg/L, respectively. The method is easy, quick, highly sensitive, and suitable for determination of betaine in sugabeet.