As a major public health problem worldwide, cancer is the top leading cause of death in China and the second leading cause of death in the United States. In recent years, the antitumor potential of natural products, especially herbs from formulas of traditional Chinese medicine (TCM), have attracted increasing attention due to their multiple targets and less side effects. Forsythiae Fructus, the fruits of Forsythia suspensa (Thunb.) Vahl, Lianqiao in Chinese, is a typical heat-clearing and detoxicating herb according to the traditional Chinese medicine theory. In our previous study, Forsythiae Fructus showed good anticancer activity. In most cases, Green Forsythia (GF) and Ripe Forsythia (RF) are used without distinction in medicinal applications. Firstly, metabolomics approach was performed to compare componential differences of two Forsythiae Fructus subtypes aqueous extracts. 15 chemical compounds were identified as specific markers to distinguishing GF and RF. Among these marker compounds, forsythoside I, forsythoside A, forsythoside E and pinoresinol were demonstrated to be key important active compounds that is account for the different anticancer efficacy between GF and RF. Next, we investigated the antitumor effect of Forsythiae Fructus aqueous extract (FAE) on B16-F10 melanoma in vivo. The transplanted B16-F10 melanoma in C57BL/6 mice were established and used for the evaluation of in vivo antitumor effect of FAE. FAE strongly inhibited the growth of B16-F10 cells in vitro and the tumor growth in vivo. The survival time of tumor-bearing mice was significantly prolonged by FAE. We further evaluated the in vivo anti-melanoma activity of Forsythiae Fructus and globally explored the serum metabolome characteristics of B16-F10 melanoma-bearing mice by UPLC/Q-TOF MS-based metabolomics study. We identified 17 metabolites which were correlated with the antitumor effect of FAE. Most of these metabolites are involved in glycerophospholipid metabolisms. These findings indicated that modulation of glycerophospholipid metabolisms may play a pivotal role in the growth of melanoma and the antitumor activity of FAE. Finally, we developed a more efficient method to fast identify active compounds with good anticancer effects in Forsythiae Fructus by integrating UPLC/MS-based metabolomics analysis and bioactivity-guided multivariate data analysis. Through the integrated metabolomics analysis, betulinic acid was predicted as top active anticancer compound in Forsythiae Fructus, and we further confirmed its good anticancer activity on B16-F10 melanoma cells. Taken together, our findings indicated that the antitumor effect of heat-clearing and detoxicating herbs involved the decreased oxidative stress and inflammation in tumor, which is closely related to their properties. Metabolomics approach play important roles in quality evaluation, efficacy evaluation and bioactive constituent identification of TCM, which is beneficial to TCM modernization.