This study presents a high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC–ESI/MS/MS) technique for the simultaneous determination of tafetinib (SIM010603) and its main metabolite (M1) in dog plasma by using Prazosin hydrochloric acid as the internal standard (IS). Both compounds were extracted from dog plasma with ethyl acetate and were separated by HPLC on a reversed phase C18 column with a mobile phase of 10 mM ammonium acetate buffer containing 0.1% formic acid–acetonitrile (40:60, v/v) at a flow rate of 0.2 mL/min. For quantification, the triple-quadruple MS was used in selected reaction monitoring (SRM) mode. The monitored transitions were m/z 425.3 → 309.2 for tafetinib, m/z 397.2 → 309.2 for M1 and m/z 384.2 → 247.1 for IS. The developed method had a short run time of 4 min and good linearity was observed over a wide range of 1–1000 ng/mL for the two compounds. The method was successfully applied in the pharmacokinetic study of tafetinib and M1 in dog.