Two-dimensional electrophoresis (2-DE) is known as the most effective as well as one of the simplest methods for separating proteins. However, a few hundred plant leaf proteins out of thousands visualized on a 2-DE gel can be identified by chemical analysis due to the presence of ribulose bisphosphate carboxylase/oxygenase (Rubisco) that limits protein loading. We describe the extraction and fractionation technique with polyethylene glycol (PEG) to analyze rice leaf proteins. Rice proteins were extracted with Mg/NP-40 extraction buffer. The Mg/Nonidet P-40 (NP-40) buffer extract was further fractionated with PEG into three fractions: 10% PEG and 10-20% PEG precipitants and the final supernatant fraction that was precipitated with acetone. Rubisco, the most abundant rice leaf protein, was enriched in the 20% PEG precipitant. This fractionation technique analyzed at least 2600 well-separated protein spots and exhibited less than 1.2% of noticeable overlapping spots. An immunological approach was used to verify the efficiency whether PEG fractionation technique can detect or enrich signal transduction components such as Ga, ADP ribosylation factor, small GTP binding protein and 14-3-3. The ADP ribosylation factor (ARF) and Ga were only detected in the PEG supernatant fraction not in the total protein fraction. The small GTP binding protein (Rab 7) was identified in the 10% PEG fraction and only faintly in the total protein fraction. The 14-3-3 protein was detected in all fractions but was especially prevalent in the 20% PEG fraction.