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A new screening approach for anticancer active compounds is presented. In this method, a target enzyme is incubated with a mixture of compounds, and then the complex formed by the target and small molecules is separated from the rest of the mixture by ultra-filtration. The complex that is retained on the membrane is subsequently washed with acid and small molecules that are specifically bound to the target are released and collected, then analyzed by high-performance liquid chromatograph combined with electrospray ionization mass spectrometry (HPLC/ESI-MS) analysis. We have successfully applied this method to screen anti-cancer compounds. DNA topoisomeraseⅡ is used as a target to capture anti-tumor candidates from a mixture of combinatorial compounds, such as doxorubicin, daunorubicin and pravastain.