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Previously compound12showed great anti‐trypanosome activity without toxicity in anin vivostudy. In the current study, a sensitive and rapid high‐performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed and validated to investigate its pharmacokinetics in mouse plasma. A protein precipitation method was applied to extract the compound, and it was then separated using a Kinetex C₁₈column with mobile phase consisting of acetonitrile–0.1% formic acid water (50:50, v/v) at a flow rate of 300 μl/min. The analytes were detected with the multiple reaction monitoring in negative electrospray ionization source for quantitative response of the compounds. Compound12was detected atm/z477.0 → 367.2, while the internal standard compound14was detected atm/z499.2 → 268.2. Inter‐ and intra‐day precision was <5.22 and 2.79% respectively, while the accuracy range was within ±9.65%. The method was successfully applied to evaluate the pharmacokinetics of compound12in mouse plasma with two formulations (20% Cremophor EL or sesame oil) and drug administration routes (oral and intraperitoneal injection). We observed a better drug serum concentration with the Cremophor formulation, and the two different drug administration routes did not show significant differences from the drug distribution.