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Botanical-based natural products are an important resource for medicinal drug discovery and continue to provide diverse pharmacophores with therapeutic potential against cancer and other human diseases. A prototype Traditional Chinese Medicine (TCM) plant extract library has been established at the US National Cancer Institute, which contains both the organic and aqueous extracts of 132 authenticated medicinal plant species that collectively represent the potential therapeutic contents of most commonly used TCM herbal prescriptions. This library is publicly available in 96- and 384- well plates for high throughput screening across a broad array of biological targets, as well as in larger quantities for isolation of active chemical ingredients. Herein, we present the methodology used to generate the library and the preliminary assessment of the anti-proliferative activity of this crude extract library in NCI-60 human cancer cell lines screen. Particularly, we report the chemical profiling and metabolome comparison analysis of four commonly used TCM plants, namely Brucea javanica, Dioscorea nipponica, Cynanchum atratum, and Salvia miltiorrhiza. Bioassay-guided isolation resulted in the identification of the active compounds, and different extraction methods were compared for their abilities to extract cytotoxic compounds and to concentrate biologically active natural products., (Published by Elsevier B.V.)