Cancer is a disorder that results in abnormal and irregular proliferation of cells in the organ or tissue of the body. In Turkey flora, there are many medicinal plants that have great therapeutic importance. Rhaponticoides iconiensis is endemic to Turkey. In the present study, the methanol extracts and sub-extracts of leafy stem and flowers of R. iconiensis were evaluated for cytotoxic activity against Colo 205, A549, HepG2, MCF7 and Beas-2b cell lines by MTT method. The compounds of R. iconiensis extracts were characterized and quantitated by LC–MS/MS. As a result, methanol extracts were not cytotoxic against A549, but were cytotoxic to the Colo 205, HepG2, MCF-7 and Beas-2b cell lines. Among the sub-extracts, dichloromethane, ethyl acetate and n-hexane sub-extracts of flowers and dichloromethane sub-extract of the stem were most cytotoxic against A549, HepG2, MCF-7 and Colo 205 cell lines, respectively. As sub-extracts were most effective against MCF-7 cancer cell line, it can be said that extracts might be effective in estrogen-sensitive cancer cells such as breast and cervix. The cytotoxic effects of sub-extracts can be explained by the variances of compounds and synergistic effects of different secondary metabolites.