Objective To establish an ultra-fast liquid chromatography with tandem mass spectrometry (UPLC-MS-MS) method for simultaneous determination of two active components in Belladonna tablets (hyoscyamine sulfate, scopoletin). Methods Chromatographic separation was performed on a Waters Acquity UPLC HSS T3(2.1 mm × 100 mm, 1.8 μm). The mobile phase consisted of 0.1% formic acid in water-methanol B was subjected to gradient elution(0~1 min 10% B; 1~4 min, 10% B→90% B; 4~6 min, 90% B; 6~6.2 min, 90%, B→10% B; 6.2~8 min, 10%B) at a flow rate of 0.2 mL·min-1. The column temperature was maintained at 40 ℃, and injection volume was 4 μL. Detection was performed with electrospray ionization source in positive and negative ionization mode. Quantifacantion was performed using multiple reactions moniroring(MRM) mode. Results The linear range of hyoscyamine sulfate and scopoletin were 193.6~968.0 ng·mL-1(r= 0.9996) and 1.00~50.00 ng·mL-1(r=0.9999) . Whose average recoveries were 94.3% and 98.0% (n=6). The RSD were 1.18% and 2.26%. Conclusion The method is simple, sensitive, accurate and efficient. It can be used for the quality control of Belladonna tablets.