Abstract: The two-dimensional gel electrophoresis（2-DE）is the most popular techniques for protein separation, and the key technology of 2-DE is the protein extraction. Plant tissues usually contain high levels of proteases and secondary metabolites that severely interfere with protein extraction, separation, and identification. Preparing the high-quality protein samples from plant tissues for proteomic analysis faces a great challenge. This article briefly describes the critical points on protein separation, especially secondary metabolites in plant tissues, and removal strategies. In addition, it also provides an updated overviews of three total protein extraction methods, their advantages and disadvantages and their applications for protein extraction from plant tissues.