An accurate, precise, selective, and sensitive high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) method was developed for the simultaneous determination of baicalin and its metabolite, baicalein 6-O-glucopyranuronoside, in normal and febrile rats plasma. Two analytes, along with hesperidin as an internal standard, were determined by multiple reactions monitoring (MRM) operated in the positive electrospray ionization (ESI) mode. Chromatographic separation was performed on an Agilent ZORBAX Extend-C18 column (100mm×2.10mm, 3.5μm) with a mobile phase of 0.1% formic acid solution and acetonitrile at a flow rate of 0.6mL/min. The calibration curves showed good linearity (r≥0.9974) with the concentration ranges of 2.000-2000ngmL for baicalin and baicalein 6-O-glucopyranuronoside. The inter- and intra-day accuracies (relative error, RE%) were between -6.62% and 6.75%, and the precisions (relative standard deviation, RSD%) were less than 9.09% for quality control samples (QCs). The method also possessed good selectivity, recovery and stability, and was successfully applied to a comparative pharmacokinetic study of baicalin and baicalein 6-O-glucopyranuronoside in normal and febrile rats after oral administration of baicalin and Chaiqin Qingning capsule.