OBJECTIVE： To establish a method for the determination of piperitylmagnolol in the incubation system of liver microsomes， and to investigate the metabolic characteristics of it in different species of liver microsomes. METHODS： The piperitylmagnolol were respectively dissolved in NADPH activated liver microsome incubation systems of human， rat， mouse， monkey and dog， and then incubated in water at 37 ℃. The reaction was terminated with methanol at 0， 2， 5， 10， 15， 20， 30， 45 and 60 minutes of incubation， respectively. Using magnolol as internal standard， UPLC-MS/MS method was used to determine the concentration of piperitylmagnolol in the incubation system. The determination was performed on Acquity UPLCTM CSH C18 column with mobile phase consisted of 0.1％ formic acid-methanol （gradient elution） at the flow rate of 0.3 mL/min. The column temperature was set at 30 ℃， and the sample size was 2 μL. The ion source was electrospray ion source， and the positive ion scanning was carried out in the multiple reaction monitoring mode. The ion pairs used for quantitative analysis were m/z 401.2→331.1 （piperitylmagnolol） and m/z 265.1→247.0 （internal standard）， respectively. Using the concentration of piperitylmagnolol at 0 min of incubation as a reference， the residual percentage， metabolism half-life in vitro （t1/2） and intrinsic clearance （CLint） were calculated for different incubation systems. The metabolic pathway of piperitylmagnolol was studied by chemical inhibitor method. Under the above chromatographic conditions， the metabolites in vitro were preliminarily analyzed by first-order full scanning and positive ion detection. RESULTS： The linear range of piperitylmagnolol was 3.91-500.00 ng/mL. The limit of quantitation was 3.91 ng/mL. RSDs of intra-day and inter-day were less than 10％. The accuracy ranged 87.40％-103.75％. Matrix effect didn’t affect the determination of the substance to be measured. The piperitylmagnolol was metabolized significantly in human， rat， mouse and dog liver microsomes， but not in monkey liver microsomes. After incubating for 30 min， residual percentage of piperitylmagnolol kept stable in different species of liver microsomes. The t1/2 of piperitylmagnolol were 12.07， 17.68， 17.59， 216.56 and 61.88 min in human， rat， mouse， monkey and dog liver microsomes； CLint were 0.115， 0.078， 0.079， 0.006， 0.022 mL/（min·mg）， respectively. Inhibitory rates of CYP2A6， CYP2D6， CYP2C19， CYP3A4， CYP2C9， CYP2E1 and CYP1A2 to compound metabolism were 55.76％， 93.94％， 96.01％， 93.69％， 71.81％， 23.25％， 28.04％， respectively. Quasi-molecular ion peaks of the two main metabolites of piperitylmagnolol in human liver microsomes were m/z 441.2（[M+Na]+） and m/z 337.2（[M+H]+）， respectively. CONCLUSIONS： Established UPLC-MS/MS method is simple， rapid and specific， and can be used for the determination of piperitylmagnolol concentration in the incubation system of liver microsomes and pharmacokinetic study. The metabolic characteristics of the compound are different among liver microsomes of human， rat， mouse， monkey and dog. Its metabolism process may be associated with CYP2D6， CYP2C19， CYP3A4， CYP2C9， etc.