Six esterase inhibitors, namely EDTA·2Na+, NaF, phenylmethanesulfonyl fluoride, dichlorvos, bis-nitrophenyl phosphate(BNPP) and thenoyltrifluoroacetone, and the mixture of NaF and BNPP were evaluated for the stabilization of zeylenone in rat plasma.The combination of NaF and BNPP was found to exhibit the most effectively stabilizing effect with the degraded content of zeylenone decreased from more than 60% (in the absence ofinhibitors) to < 6 %.Following the stabilization by the addition of NaF(5 mM) and BNPP (5 mM), the analyte and the internal standard (IS), emodin, in rat plasma were acidified by formic acid and extracted into ethyl-acetate at 0℃.After chromatographic separation, the detection of zeylenone was performed on a 3200 Q-Trap with positive ion electrospray mode, monitoring the ion-transition m/z 383.2→105.0.The method was validated over the range from2 to 1300 ng/mL with inter-and intra-run precision for the quality control samples being less than 6.8%.The assay accuracy was within100 ±7.0% of the nominal values.The validated method was applied to a pharmacokinetic study in rats after the administration of zeylenone solutions via intratracheal route.The results demonstrated that the assay was adequately sensitive, stable, rapid and specific for the analysis of plasma samples in preclinical pharmacokinetic studies.