Rosemary bioactivity is correlated with the presence of phenolic diterpenes (carnosic acid, carnosol) and phenolic acids (rosmarinic acid). In the present work, two processes, pressurized liquid extraction (PLE) and supercritical antisolvent fractionation (SAF) were integrated to separate and enrich these families of compounds. The optimization of the SAF process was carried out using a response surface methodology (RSM) with three factors: (i) CO2 pressure, (ii) percentage of water in the PLE extract and (iii) PLE extract/SC-CO2 flow ratio. The selected responses were the relative amount of each compound (rosmarinic acid (RA) and camosic acid + carnosol (CA+ CS)), total phenolic content, antioxidant activity and antiproliferative activity against HT-29 colon cancer cells. The higher CA + CS content was obtained at 100 bar, 50% (v/v) of water in the feed solution and 0.025 feed/SC-CO2 mass flow ratio, achieving values of 478.1 mg/g extract and 17% of cell survival after 24 h of treatment. (C) 2015 Elsevier B.V. All rights reserved.