Abstract: The higher content of high-abundance proteins (Ribulose-l, 5-bisphosphate carboxylase/oxygenase; RuBisCo) interferes with the dynamic resolution of proteins in two-dimensional electrophoresis (2-DE), affecting the detection and identification of functional proteins in proteomics. To explore suitable methods for removing high-abundance protein, Mg/NP-40 and different concentrations of polyethylene glycol (PEG) were used for protein pre-fractionation and the treatments’ influence on the separation of proteins in alfalfa leaf was compared. The results indicated that (335±17), (417±3), (445±7) and (459±11) spots were detected in treatments of 0, 15%, 17.5% and 20% PEG respectively. There were significant differences between the 0-17.5% treatments but no significant differences were found between the 17.5% and 20% treatments. More differential protein spots were detected in the 17.5% treatment, which thus proved the most effective way of removing RuBisCo proteins. In order to inspect the applicability of this result, a proteomic study of alfalfa in response to low temperatures was under taken. Compared with total proteins extracted by TCA/acetone, eight new protein spots were identified after PEG treatment to remove high-abundance proteins. The research thus indicates that pre-fractionation with Mg/NP-40 and17.5% PEG is suitable for proteomic studies of alfalfa.