Tetrandrine (TET) was approved by the China Food and Drug Administration (CFDA) for the treatment of silicosis. However, patients can’t use this effective drug chronically due to side effects such as hypersomnia, asthenia, etc. The purpose of this study is to develop an UPLC–MS/MS method to quantify TET and its major metabolite and apply the method in a single dose human pharmacokinetic study. A Restek UItra BiPh column (100 × 2.1 mm, 5 μm) was used with acetonitrile and 0.1% formic acid in water as the mobile phases. The mass analysis was performed in a Waters Xevo TQ mass spectrometer via multiple reaction monitoring (MRM) with positive scan mode. A one-step protein precipitation by acetonitrile was used to extract the analytes from blood sample. The method showed linearity in the concentration ranges of 2.05–1050.00 ng/mL for TET and 1.27–650.00 ng/mL for berbamine. The intra/inter-day precisions were less 15% for these two analytes. The extraction recoveries of these two analytes were from 75.6% to 107.8% and the matrix effects ranged from 92.4% to 110.4%. The stabilities of these compounds in plasma were evaluated by analyzing three different concentrations following storage at 25 °C for 6 h, and −80 °C for 30 days. All the samples displayed less than 15.0% variations. The validated method was applied to PK study in human and the PK parameters of TET and berbamine were determined. In conclusion, a robust and sensitive LC–MS/MS method was developed and validated. In addition, the results of human PK experiment showed that TET and berbamine could be accumulated and more study is needed to establish a reasonable dose segment.