In this paper, the authors present the obtained results on mitochondrial protein extraction with high yield and quality for two dimensional spestrophoresis (2DE) analysis of Ice plant (Mesembryanthemum crystallinum L.). The leaves of Ice plant were used for isolating the mitochondria via diferent centrifugation and time at 4°C. Washed mitochondria were further purified by Percoll gradient at different concentrations. the authors used phenol solution to extract mitochondrial purified protein, and then the authors determined the different protein on 2DE gels. The obtained results on mitochondrial protein content as well as the protein expression from 2DE gels showed that using phenol to extract mitochondrial protein leaded to higher yield and quality than previous methods which were used with other CAM plants. The method from this paper can be use to extract total protein in mitochondria with high yield and quality, suitable for further study on mitochondrial proteomic analysis in order to exploit the significant proteins which were attended and operated in metabolism of plant cells.