A rapid, sensitive and reliable quantitative method based on ultra-high performance liquid chromatography coupled with Q-Exactive Orbitrap tandem mass spectrometry was developed for simultaneous determination of berberine, berberrubine, palmatine, jatrorrhizine, columbamine, baicalin, baicalein and wogonin in rat plasma after oral administration with Yan-Ke-Ning-Tablet (YKNT). After precipitation with acetonitrile, the plasma samples were separated on a reverse-phase C-18 column with 1 mm ammonium acetate containing 0.2% acetic acid-acetonitrile as mobile phase. Calibration curves showed good linearity (r > 0.9983) over the tested concentration ranges of 0.5-200 ng/mL for berberine, berberrubine, palmatine, jatrorrhizine and columbamine, and 1-300 ng/mL for baicalin, baicalein and wogonin. The precision (relative standard deviation) at three different concentration levels was <12.15% and the accuracy (relative error) ranged from -8.24 to 10.85%. No matrix effects were observed with matrix effect value ranging from 89.23 to 107.68%. The extraction recovery was in the range of 82.34-92.31%. The validated assay was further successfully applied to the pharmacokinetic study of these components after oral administration of YKNT. The present study provides the pharmacokinetic profiles of major bioactive components found in YKNT, and provides valuable information regarding the chemical components that were absorbed into plasma, which will be helpful for understanding the therapeutic effects of YKNT.