ABSTRACT: Objective Hedyotis corymbosa (Shui Xian Cao, 水线草, HC) is the main antitumor medicine in China and other southeast Asian countries. A comprehensive quality control system of HC was established in this study, as the current system was ineffective.Methods Microscopy was used to observe the characteristics of the powder and cross-sectional tissue. Thin layer chromatography (TLC) was employed to separate HC from Hedyotis diffusa (Bai Hua She She Cao, 白花蛇舌草, HD). Simultaneously, high-performance liquid chromatography (HPLC) was used to establish the characteristic spectrum of HC and determine the concentrations of hedyotiscone A. Regarding the issue of systematicness, concentrations of water, total ash, acid-insoluble ash, heavy metals (including lead, arsenic, mercury, cadmium and copper) and pesticide residue were measured by the methods described in the 2015 edition of Chinese Pharmacopoeia.Results Calcium oxalate needle crystal bundles and oil drops were observed in the cross-sectional tissue. Three characteristic blue fluorescent spots were observed in the HC samples by TLC. There were five distinct characteristic peaks in HC but not in HD, and hedyotiscone A was the sharpest characteristic peak. The generated linear equation of hedyotiscone A was Y = 3.15 × 108X + 312.04 (r2 = 1.000) between 0.00005270 and 0.002108 mg/mL, and the average recovery was 100.42%. Precision [relative standard deviation (RSD) 0.92%], repeatability (RSD 0.14%), and recovery (RSD 0.85%) met the requirements for quantitative analysis. Hedyotiscone A concentrations ranged from 0.00017%∼0.00113% across different habitats. Concentrations of water, ash and acid-insoluble ash were 8.80%∼11.2%, 7.00%∼15.3% and 0.440%∼7.70%, respectively. Concentrations of Pb, As, Hg, Cd and Cu were 1.60∼4.60, 0.650∼1.60, 0.0300∼0.100, 0.240∼0.690 and 10.1∼16.6 µg/g, respectively. Pesticide residues were less than 0.00100 ppm.Conclusion The quality control system can effectively distinguish HC from HD, so as to comprehensively control the quality of HC, to provide references for the clinical use of HC. Keywords: Hedyotis corymbosa, Hedyotis diffusa, Hedyotiscone A, TLC, HPLC, Quality standard