Paeonol, a principal bioactive component of the Chinese herb Moutan Cortex with anti-inflammatory and analgesic effects, was comparatively studied to determine its pharmacokinetic behavior and metabolic profile in rat following oral administration of the pure paeonol alone and an herbal preparation "Qingfu Guanjieshu" (QFGJS) containing paeonol. An HPLC-DAD method was developed and validated for determining the concentration of paeonol in rat plasma. The in vivo time curves and AUC of paeonol at three doses were increased in a dose-dependent manner, while the pharmacokinetic parameters of paeonol in QFGJS at a comparable dosage were significantly elevated in comparison with those of pure paeonol. By using LC-Q/TOF-MS technique, four metabolites of paeonol were identified in plasma at 5min after dosing, with T(max) around 20min after treatment with the pure paeonol or QFGJS. Interestingly, relative concentrations of metabolites P2, P3 and P5 were markedly increased in plasma of rats treated with QFGJS compared with those of pure paeonol. These results indicate that other components in QFGJS could effectively influence the pharmacokinetic behavior and metabolic profile of paeonol in rats. The current studies emphasize the significance of the research toward an understanding of pharmacokinetic interactions of the co-existing components in the herbal preparations.