To isolate high molecular weight (HMW) or low-abundance proteins we exploited the high resolving power provided by the molecular sieves of polyacrylamide gel matrices. Rice-leaf protein extracts were applied to a single well of an SDS-polyacrylamide gel with prestained molecular size markers at both ends. After electrophoresis, the gel was cut into 4 segments according to size, and each segment was ground in extraction buffer. The eluted proteins were separated from the gel matrix by centrifugation followed by acetone precipitation, and the precipitated proteins were subjected to SDS-PAGE and 2-DE. The SDS-PAGE-based prefractionation method provided non-overlapping discrete sample pools. About 27% more protein spots were detected in the fractionated samples than in the unfractionated samples, and 17% were enhanced. The improvement was especially prominent in the case of HMW proteins. Well-separated HMW proteins were analyzed by MALDI-TOF mass spectrometry. The molecular masses of the identified proteins in the > 48 kDa gel segment were distributed between 50 and 112 kDa, thus validating this prefractionation method. Identified HMW proteins with similar mass but different pI were mostly isoforms. Thus SDS-PAGE-based size prefractionation provides improved separation and detection of HMW proteins.