An UPLC-MS/MS method was developed for the quantification of afatinib and paclitaxel in rat plasma.The method was also applied in the investigation on the pharmacokinetics of the analytes.The plasma samples were extracted by solid-phase extraction(SPE),and seperated on an Agilent Zorbax Eclipse Plus C18(100 mm×2.1 mm，1.8 μm) column with acetonitrile and water containing 0.1% formic acid as mobile phases at a flow rate of 0.4 mL/min,a column temperature of 40 ℃ and a sample injection of 3 μL.The quantification analysis was performed by MS with positive electrospray ionization(ESI+) source in multiple reaction monitoring(MRM) mode.The calibration curves for two analytes were linear over the concentration range of 0.5-1 500 μg/L,with correlation coefficients(r2) of 0.998 and the lower limits of quantitation(LLOQs) of 0.5 μg/L.The inter-day RSDs and intra-day RSDs were not more than 8.4%,and the accuracy ranged from 92.5% to 107.1%.The average recoveries were in the range of 86.2%-95.1%.The matrix effect values were sufficiently minimized to 86.6%-108.6%.The validated method is rapid and sensitive.It could be applied in the determination of afatinib and paclitaxel in plasma,and provide an analysis means for the clinical medication adjustment.