In this study， a simple and sensitive quantitation method based on liquid chromatography combined with diode array detector and Q-Exactive-Orbitrap tandem mass spectrometry was developed for the determination of MK-8353 in rat plasma. The chromatographic separation was carried out on a Waters ACQUITY BEH C column by using water containing 1?mM ammonium acetate and acetonitrile containing 0.1% formic acid as mobile phase. The developed assay was linear (r > 0.999) over the concentration range of 1-1000?ng/mL. The selectivity， precision， accuracy， recovery， matrix effects and stability were all within the required limits. The validated assay has been further applied to the pharmacokinetic study of MK-8353 in rat after intravenous and oral administration， which revealed that MK-8353 showed low clearance and satisfactory bioavailability. More importantly， the metabolites of MK-8353 present in rat plasma， RLM， DLM and HLM were identified and profiled. Under the current conditions， a total of 10 metabolites were detected and their chemical structures were proposed in terms of the accurate masses and their fragment ions. Our results revealed that MK-8353 was metabolized mainly through dealkylation， demethylation， depropylation， oxygenation， sulfur oxidation and formation of lactam. Compared with animal species， no human-specific metabolite was found in HLM. This study provides overall in vitro and in vivo profiles of MK-8353， which is of great help in understanding its PK/PD profiles and in predicting human pharmacokinetic profiles.