Objective:This study aimed to establish a new method for the determination of matrine and oxymatrine in honey by UPLC-MS/MS. Methods: The honey was dissolved in water, and the liquid-liquid extraction pretreatment technology was adopted. The extraction was achieved using ammonia-acetonitrile, with the addition of sodium sulfate to induce a salting out effect. Chromatographic separation was achieved on Inspire HILIC column in the mobile phase composition of acetonitrile and 0.1% formic acid5 mmol/L ammonium acetate buffer. Mass Spectrometry was carried out under the multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI+) and external standard method for quantitative analysis. Results: The linear relationship between matrine and oxymatrine was observed in the range of 1~100 ng/mL, with correlation coefficients higher than 0.998. The detection limit and quantitative limit of matrine and oxymatrine in honeywere 0.1 μg/kg and 0.3 μg/kg, respectively. The average recoveries of matrine and oxymatrine were 90.9%~95.1% and 80.9%~84.3%, respectively, at the spiked levels of 1, 10 and 100 μg/kg, with the relative standard deviations (RSDS)＜ 10% (n=6). The validated method was then successfully applied to the determination of matrine and oxymatrine in 45 commercially available honey, and the results showed that the detection probability and content of matrine and oxymatrine in acacia honey were relatively high. Conclusion: The established method is accurate, rapid, and sensitive, Moreover, a simultaneous preparation of multiple sample extracts is available, and it can used for the routine determination of matrine and oxymatrine in honey.