As a well-known Chinese folk medicine, Prunellae Spica is widely used in medical treatment,functional beverage and soup in many provinces of China and the East Asian regions.In order to control its quality better, a comprehensive analytical method was developed for the simultaneous determination of 21 bioactive constituents including triterpenoids, phenolic acids, flavonoids and coumarins in Prunellae Spica by high performance liquid chromatography coupled with triple quadrupole-linear ion trap mass spectrometry (HPLC-QTRAP-MS/MS).The separation was carried out on an Syergi Hydro-RP C18 columa (100 mm × 2.0 mm, 2.5 μ m) with 0.1% formic acid aqueous solution and methanol as the mobile phase under gradient conditions.The target compounds were analyzed by the negative ion multiple reaction monitoring (MRM) mode.The developed method was validated in terms of accuracy, precision, stability, linearity and sensitivity, which was successfully applied to analyze twenty-three Prunellae Spica samples collected from different regions in China and four Prunellae Spica samples gathered at different growth periods.Furthermore, hierarchical cluster analysis (HCA) and grey relational analysis (GRA) were performed to evaluate these samples from different habitats and different growth periods according to the contents of 21 constituents.This study might provide valuable information for comprehensive evaluation of Prunellae Spica quality and scientific selection of its optimal harvest period.