The kaurenoic acid-type diterpenoids in Acanthopanacis Cortex have been reported to be the major active components. However, the diterpenoids are present as position isomers that exacerbate the challenges in obtaining standards compounds. Little work has been done on the quantitative analysis of the diterpenoids in the herb. In the present study, two diterpenoid isomers ent-16 βH,17-isovalerate-kauran-19-oic acid ( 1) and ent-16 βH,17-methyl butanoate-kauran-19-oic acid ( 2) with high purity were separated by analytical HPLC, followed by recrystallization in acetone. Furthermore, an HPLC-ELSD method was developed and validated for simultaneous determination of 1 and 2 in 9 batches of Acanthopanacis Cortex samples. The HPLC separation and quantification was achieved in 40 min using an Agela Promosil C 18 column eluted with a gradient of water and acetonitrile. The calibration curves showed good linearity ( r 2 ≥ 0.999 9) within the test ranges. The LOD ranged from 0.407 2 to 0.518 0 μg and LOQ ranged from 1.018 0 to 1.295 0 μg. The precisions (%RSD) were within 1.47% for the two isomers. The recovery of the assay was in the range of 98.78%–99.11% with RSD values less than 2.76%. It is the first time to establish a quantitative HPLC method for the analysis of the bioactive kaurenoic acid isomers in the herb.