Background:Little information is available about chemical components of the Cousinia genus. A primary cytotoxicity screening on Cousinia verbascifolia showed moderate cytotoxic activity against OVCAR-3 ovarian and HT-29 colon cancer cells. Therefore, the aim of this study is a phytochemical investigation to identify the compounds responsible for this bioactivity.Materials and Methods:Extraction was done through percolation and fractionations by reverse phase column chromatography and normal column chromatography. Using standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay Fr.b8 with moderate cytotoxicity was selected for identification of major components. Fr.b8 was subjected to polyamide column chromatography. More purification was done using a new modified recycle high-performance liquid chromatography (HPLC) with flow splitter.Results:Two known compounds: Apigenin (flavone) and caffeic acid (phenolic acid) were obtained from phenolic bioactive fraction for the first time from this plant.Conclusions:Apigenin and caffeic acid with known antitumor and matrix metalloproteinase inhibitory effects seem to be the bioactive components responsible for moderate cytotoxicity of phenolic fraction. Recycle HPLC following with flow splitting is a new method useful for isolation of closely eluted compounds in HPLC chromatogram.