An improved method for extracting proteins from leaf tissues of Aloe vera L., a recalcitrant plant species, for proteomic analysis is presented. In this protocol, the following critical components are included. A washing step is added prior to homogenization of the tissue to eliminate contaminants, and a concentrated 2× extraction buffer (pH 7.5) is used to increase protein yield. Compared to classical trichloroacetic acid-acetone and phenol extraction methods, this novel protocol has yielded two-dimensional electrophoresis gels with minimal (if any) streaking and provided high-quality protein samples. This protocol is expected to be applicable to other recalcitrant plant tissues.