Background: Rabdosia rubescens is a traditional herbal medicine, commonly known as an anti-cancer plant. A large number of literatures have reported that the diterpenoids in R. rubescens have strong anti-tumor effects. The purpose of this study was to isolate the anti-cancer active ingredients from R rubescens, identify their structures by nuclear magnetic resonance (NMR), and establish high-performance liquid chromatography (HPLC) method for the determination of content of the oridonin and the ponicidin in R. rubescens. Materials and Methods: The anti-cancer components of R. rubescens were isolated using silica gel column chromatography, and their structures were identified by NMR technology. HPLC method was established for the determination of the content of the oridonin and the ponicidin in R. rubescens collected from July to October in Taihang Mountain, China. Results: Two anti-cancer components—the oridonin and the ponicidin—were isolated and identified. HPLC methods for the determination of the oridonin and the ponicidin were established. This method could be used to complete the chromatographic analysis of the oridonin and the ponicidin within 10 min, and the chromatographic peaks of each component reached the baseline separation. The contents of the oridonin and the ponicidin in the whole grass of R. rubescens collected from July to October were 0.469%, 0.618%, 0.625%, 0.448% and 0.124%, 0.203%, 0.216%, 0.127%, respectively. The results showed that August to September was the best harvest time, and the contents of the oridonin and the ponicidin were the highest during this time. The specific harvest time was determined by the climate and precipitation of the year. Conclusion: The HPLC method established in this study was simple, rapid, accurate, reliable, and reproducible, which provided a reference for drug application and resource utilization of R. rubescens and could be used for quantitative analysis of anti-cancer active ingredients in R. rubescens.