Abstract: To establish a quantitative LC-MS/MS method for the simultaneous detection of components of Erlong Zuoci Pill in rat plasma: verbascoside, oxypaeoniflorin, echinacoside and benzoylpaeoniflorin, and to evaluate the pharmacokinetic characteristics of Erlong Zuoci Pill in rats, plasma samples were purified by protein precipitation using methanol as a protein precipitant.Methanol was used as the organic phase and aqueous solution containing 0.1% formic acid was used as the water phase.The quantitative analysis method of verbascoside, oxypaeoniflorin, echinacoside and benzoylpaeoniflorin was established in negative ion mode, and the validation of bioanalytical method was carried out.Healthy SD rats were selected, and 20 mL/kg (equivalent to the original drug 10 g/kg dose) of Erlong Zuoci Pill extract was administered by intragastric administration.The plasma concentration of the target compounds at different time intervals after administration was determined using the established method, and the pharmacokinetic parameters was calculated by the Phoenix WinNonlin8.3 software using the non-compartmental model.The method validation results showed that verbascoside (r = 0.993 7) and oxypaeoniflorin (r = 0.994 6) had good linear relationship in the concentration range of 0.5-50 ng/mL, echinacoside (r = 0.993 6) and benzoylpaeoniflorin (r = 0.992 6) had good linear relationship in the concentration range of 1-100 ng/mL.The relative standard deviations of the inter- and intra- batch precision of the four compounds were all less than 15%, and the inter- batch and intra- accuracies were between 85% and 115%.Extraction recovery, matrix effect and stability met the relevant requirements.After a single gavage of Erlong Zuoci Pill extract in rats, all the four compounds were rapidly absorbed and eliminated.Oxypaeoniflorin, echinacoside, and benzoylpaeoniflorin showed two peaks in their drug concentration-time curves.Compared with the other three compounds, oxypaeoniflorin has the highest concentration in rat plasma with c_max1 of (24.40 ± 4.78) ng/mL and c_max2 of (22.50 ± 2.70) ng/mL. The results show that the validation results of this method are in line with the guiding principles of biological sample analysis methods, and it can be used to evaluate the pharmacokinetic characteristics of Erlong Zuoci Pill extract in rats.