Comparative proteomic analysis of chloroplast by 2-DE has received significant attention in recent years. However, the complication of membrane systems in chloroplast made it challenging to elucidate entire chloroplast proteome by 2-DE. Here, we developed an efficient method for extracting chloroplast proteins, and produced excellent 2-DE profiles from both Arabidopsis thaliana and Salicornia europaea. Comparison of this method with another two protocols for the extraction of A. thaliana chloroplast proteins showed that our method obtained higher protein yields and produced more protein spots on both pH 3-10 and 4-7 2-DE gels. Moreover, this method recovered more proteins in the basic and high M(r) regions, thereby offering the best extraction of chloroplast proteins. Identification of 15 specific chloroplast-targeted proteins on our gels by MALDI-TOF MS revealed that this method was compatible with MS, and recovered more chloroplast membrane proteins than the commonly used methods. This protocol is expected to have a wide application in future chloroplast proteomic analysis.