A novel method, HPLC-MS/MS was developed to qualitatively identify and quantitatively determine the 28 components including 19 diterpenoids, 6 phenolic acids and 3 flavonoids in Isodon rubescens, an important traditional Chinese medicine. The separation was performed on a C(18) column with linear gradient elution with 0.1% aqueous formic acid/methanol containing 0.1% formic acid at a flow rate of 0.7 mL/min. The identification and quantification of those analytes were achieved on a hybrid quadrupole linear ion trap mass spectrometer. Multiple-reaction monitoring scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. Full validation of the method was carried out (linearity, precision, accuracy, LOD and LOQ). The results indicated that the method was simple, rapid, specific and reliable. The proposed method was successfully applied for the qualitative and quantitative analysis of 28 chemical compositions in 21 batches of natural and cultured I. rubescens samples from different sources which had great variation on the contents. The results demonstrated that the method was useful for standardization and differentiation of large numbers of similar samples.