Flavonoids from Opuntia ficus-indica (L.) Mill were extracted using supercritical carbon dioxide (SC-CO2) technique. A central composite design was used to evaluate the influence of pressure, temperature and flow rate on the total extract yield and the isorhamnetin recovery. Isorhamnetin-3-O-glucosyl-rhamnosyl-rhamnoside, isorhamnetin-3-O-glucosyl-rhamnosyl-pentoside, isorhamnetin-3-O-glucosyl-rhamnoside, isorhamnetin-3-O-glucosyl-pentoside were the most abundant flavonoids in O. ficus-indica supercritical extracts. Pressure had a significant effect on the extraction yield and isorhamnetin recovery, particularly a quadratic response was observed for extraction yield. A similar flavonoids profile than the observed for the methanolic extract or after alkaline hydrolysis was obtained with the following conditions: 50 degrees C, 300 bar and a CO2 flow rate of 80 Orlin. SC-CO2 technique allowed the preferential extraction of 3-O-methyl-quercetin at 400 bar, 35 degrees C, and a flow rate of 100 gimin. This report shows the potential uses of SC-CO2 as an alternative solvent free methodology to extract high value ingredients from natural sources. (C) 2016 Elsevier B.V. All rights reserved.