Abstract: Three protein extracting methods (Tris-phenol method, TCA/ acetone method and TCA/ acetone + Tris-phenol method) were compared with the materials of P. tomentosa buds and described in this report. To determine which protein extracting method is most suitable for P. tomentosa buds, two-dimensional electrophoresis was applied to the separation of proteins, PDQuest software and mass spectrometry were used to characterize the protein spots from 2D-gel. Among the three protein extracting methods, TCA/ acetone method was the most suitable one for P. tomentosa buds and yielded (3.57 ±0.618) mg/ g total protein amount in fresh sample, 231 ±4 protein spots on the 2D-Gel and the clearest 2D-Gel images, which were higher in total amount, more in spot number and clearer in 2D-Gel image than other two methods.