In this study, a high performance liquid chromatography (HPLC) coupled with diode array detection (DAD) for simultaneous determination of six flavones including baicalein, sophoricoside, rutin, baicalin, quercetin and genistein in rat plasma and tissues after oral administration of JiangYaBifeng (JYBF) tablets was developed. The investigated analytes in plasma and tissues were extracted and purified with liquid–liquid extraction and solid phase extraction (SPE). Chromatographic separation was accomplished on a DIONEX Acclaim C18 column (250 mm × 4.6 mm, 5.0 μm particle size) with a simple linear gradient elution. The calibration curves for all the flavones had good linearity in the measured range with R2 higher than 0.9983. The relative errors (REs) of the intra- and inter-day accuracy at different flavones levels were all less than ±10%. The proposed method enables unambiguous identification and quantification of investigated flavones in vivo. This is the first report on determination of the major flavones in rat plasma and tissues after oral administration of JYBF tablets. The results provided a meaningful basis for evaluating the clinical application of this medicine.