Centrifugal partition chromatography was successfully applied in the separation of close Rf complex anticancer triterpenes directly from a fraction of Eucalyptus hybrid (Myrtaceae). The experiment was performed with a two-phase solvent system composed of hexane/ethyl acetate/methanol/water (1:2:1.5:1v/v/v) where 2% ammonia solution was added in lower aqueous mobile phase to achieve pH 9.5. From 1.5g of a fraction, 145mg of ursolic acid and 72mg of ursolic acid lactone were obtained in 95.4% and 94.8% purities. The total yield recovery was 94% and the isolated triterpenes were characterized on the basis of their 1H, 13C-NMR, and ESI-MS data.