Abstract: Objective: A method was developed for the determination of arecoline, arecaidine, guvacoline and guvacine hydrochloride in areca nut seeds by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Methods: The sample were extracted with 45% ethanol aqueous solution, and detected by UPLC-MS/MS method. Chromatographic conditions: Separated by Waters Atlantis T3 (2.1 mm×150 mm, 5 μm) column, eluted by gradient with 0.1% formic acid aqueous solution-acetonitrile solution as mobile phase, with flow rate of 0.5 mL·min⁻¹, column temperature of 40 ℃, injection volume of 1 μL. Mass spectrometry conditions: Using electrospray ion source (ESI⁺), detection method multiple reaction monitoring (MRM) mode scanning, the quantitative analysis by external standard method. Results: In the range of 50~500 ng/mL, the mass concentration and peak area of arecoline, arecaidine, guvacoline and guvacine hydrochloride showed a good linear relationship (R²>0.99), and the sample recovery rates were all within the specified range of 70%~120%, the average recovery rate was 75.27%~96.70%, and the RSD was less than 7%. Conclusion: The method was good repeatability, stability, and instrument precision.