For the rational utilization and the quantitative quality control of the Stephania yunnanensis Lo, an HPLC‐DAD method was developed for the quantitative and simultaneous determination of five alkaloids in rat plasma (stepharine, sinomenine, palmatine, isocorydine and  tetrahydropalmatine), which were the main active chemical
constituents of this plant and belong to four kinds of isoquinoline‐type alkaloids (protoberberine, morphine, aporphine and protaporphine alkaloids). The contents of five alkaloids ranged from 0.09 to 2.32% (w/w). The method validation was tested for the linearity (r2 > 0.9975), precision (intra‐day RSD < 4.8% and inter‐day RSD < 4.9%), extraction recovery (85.49 ± 2.29% to 99.21 ± 1.48%) and stability (98.5 ± 5.3% to 101.2 ± 3.4%). We developed an HPLC‐DAD method to simultaneously measure these alkaloids in rat plasma after oral administration of the extract of this plant to rats. The results supported the hypothesis that isoquinoline alkaloids were the compounds responsible for the main pharmacological activities for anti‐inflammatory and analgesic.