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To investigate the herb–drug pharmacokinetic interaction of artificial calculus bovis (ACB) with diclofenac sodium (DS) and chlorpheniramine maleate (CPM) in rats.A sensitive high-performance liquid chromatography coupled with tandem mass spectrometry method was developed and validated for the simultaneous determination of DS and CPM in rat plasma. The proposed method was successfully applied to compare the herb–drug pharmacokinetic interaction of ACB with DS and CPM in rats following intragastric administration.The proposed method had good linearity and no endogenous material interfered with the analytes and internal standard peaks. The lower limit of quantification of DS and CPM was 1 and 0.1 ng/ml, respectively. There was no apparent pharmacokinetic interaction between DS and CPM. Co-administration of ACB with DS noticeably increased the area under the concentration–time curve (AUC0-∞) and peak plasma concentration (Cmax) of DS, while the parameters time of peak concentration (Tmax), clearance (ClZ/F) and apparent volume of distribution (VZ/F) of DS significantly decreased. Meanwhile, co-administration of ACB with CPM noticeably increased the Tmax, ClZ/F and VZ/F of CPM. A marked decline in AUC0-∞ and Cmax of CPM occurred in the presence of ACB.This study indicated that co-administration of ACB with DS and CPM can result in an apparent herb–drug pharmacokinetic interaction in rats.