Cytoplasma is the cell interior place between the cellular membrane and the nucleus, where various intracellular activities take place, including energy production, reactive oxygen species (ROS) detoxification, heme synthesis, nitrogen and lipid metabolism, phosphorylation in signal transduction, and cytoskeletal meshwork construction. The rich cytoplasmatic proteins carrying out these intracellular functions are interesting targets for biochemical and molecular biological studies. The relatively recent discipline of proteomics offers a chance to globally analyze the changes in cytoplasmic proteins corresponding to drug treatments or disease conditions, and thus provide target candidates for further biological validation in drug development and biomarker discovery. Isolation of cytoplasmic proteins from cells is a necessary step for high resolution protein separation by two-dimensional gel electrophoresis (2DE) and specific proteomic analysis.