Abstract Background To promote herbal medicine depends largely on its quality. Chromatographic fingerprint is a frequent approach for quality assessment of herbs however with challenges on robust and reproducibility. To develop rapid, cheap and comprehensive measurements as complementary tools for herbal quality control are still urgently needed. Moreover, biological activities are essential for herbal quality, and should be taken into consideration with emphasized in quality control. Methods In this research, HPLC fingerprint and delayed luminescence (DL, a rapid and systematic tool) were used to measure the rhubarb samples of multiple species. Statistics were explored to classify these rhubarb samples using data obtained from two analytic methods. In addition, DL properties were linked to specific chemical components which may reflect bioactivities of rhubarb using Spearman’s rank correlation. Moreover, mice model was used to evaluate the cathartic effect between rhubarb samples stratifying by two analytic methods. Results We found that there was no significant difference of chemical fingerprints and DL signals among the different species of medicinal rhubarb. However, our results show a high similarity between HPLC fingerprint analysis and DL measurements in classification of these rhubarb samples into two sub-groups. In addition, the two sub-groups of rhubarb samples that may have different cathartic activities. Conclusion This approach provides new leads for development of herbal quality assessment based on bioactivity. In conclusion, integrated assessment by measuring HPLC fingerprint and DL with emphasized on bioactivity may provide novel strategy for herbal quality control.