Feasibility of using Cyanex 301, as the auxiliary agent, for supercritical extraction of Cupper and Manganese cations from solid matrix was studied statistically. The amount of extraction is influenced by several parameters, such as amount of ligand, pressure, temperature, SCCO2 flow rate, time of extraction and amount of acid. Recent researches showed that factorial design is an effective tool for optimization of the operational conditions. A 26-3 fractional factorial design was used to derive the most effective parameters affecting the yield of extraction. Using analysis of variance and pooling method, the significant parameters were determined and separated from insignificant factors, and then main effects and their binary interactions were obtained. It was concluded that at high pressure (above 200 bar), the amount of acid, temperature and CO2 flow rate have significant effect on Cu extraction. The amount of the ligand showed positive effect on Mn extraction. As a result, it is possible to separate Cu2+ from Mn2+ by SCCO2 at appropriate conditions from solid matrix more than 95%.