AbstractSample preparation is crucial for extraction and higher resolution of proteins by two‐dimensional gel electrophoresis (2‐DE). In this study, we present an efficient protocol to extract proteins from mature rice leaves by minimizing the presence of nonprotein contaminants and by maximizing contact between the sample and extraction buffer. A combination of chemical and physical processes remarkably improved protein extraction for 2‐DE. The efficiency of this protocol was demonstrated by comparison of the rice proteome at two developmental stages.