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Proteomic study in litchi (Litchi chinensis Sonn.) has been of increasing interest in recent years. Agriculturists expect to gain better knowledge for solving the problem of fruit production caused by climate changes by proteomic studies. However, very limited information has been available due lack of a proper technique for protein extraction. Litchi plants contain a high level of secondary compounds (phenolics), which strongly reduce the purity of protein yields. This study aimed to develop a new highly efficient protein extraction technique by improving the available methods: (A) Trichloroactic acid/acetone; (B) Homogenization buffer/phenol; and (C) Phenol/SDS buffer. The results showed that method C gave the highest protein yield and resolution of protein separation using sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) and two−dimensional gel electrophoresis (2−DE), while the method A could not detect any protein and method B gave low protein separation.