Codonopsis pilosula ("Dangshen"), a traditional Chinese tonic medicine widely used as a substitute for Panax gingseng, has been the subject of chemical ingredient investigations, but few methods for the determination of its active compounds have been reported. This paper describes a reversed-phase high-performance liquid chromatographic (RP-HPLC) method for the separation and determination of 8β-hydroxyasterolid (A) and perlolyrine (B). 8β-Hydroxyasterolid is the first sesquiterpenoid lactone isolated from C. pilosula and exhibits antiinflammatory properties, while perlolyrine is the first alkaloid from C. pilosula, a β-carboline alkaloid acting on benzodiazepine and GABA receptors, and present in both C. pilosula and P. gingseng (a rare similar ingredient between the two). Different HPLC conditions were designed for A and B due to their significant content differences in samples. Calibration graphs for A (160–800 ng) and B (26–78 ng) showed good linearity, with recoveries of 101.6% and 101.0%, respectively. Content analysis of 10 Codonopsis samples (6 cultivated, 4 wild) revealed significant A variations: wild C. subglobosa and C. tangshen lacked A, while C. pilosula var. modesta from Lanping had the highest A. For B (11 samples including 1 P. gingseng), P. gingseng had higher B than most Codonopsis, some Codonopsis samples lacked B, and same species from different sources showed variations. Similar content variations of A and B are useful for quality control of Codonopsis.