A high performance liquid chromatography with electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) method has been developed to quantify 17 bioactive compounds in Sarcandra glabra and its preparation simultaneously. These constituents including flavones, coumarins, caffeoyl derivatives and sesquiterpenoids were detected in the negative ion mode ESI-MS and quantified by multiple reaction monitoring (MRM). All 17 constituents were separated and determined within 17 minutes. The linear regressions were acquired with r(2) > 0.9990, respectively. The precision was evaluated by intra-and inter-day tests, and relative standard deviation (R. S. D.) values were reported within the range of 0.86-2.29%, 1.16-4.83% and 0.87-4.94%, 0.69-4.92% for mixed standard solutions and extract solutions, respectively. The recovery studies for the assayed constituents were observed over the range of 80.20-119.68%. It is demonstrated that the method developed was successfully applied for quantification of 17 main constituents, which provided a new basis for overall assessment on the quality of S. glabra and its preparation.