General procedures for the extraction of Citrus leaf proteins for analysis by two-dimensional electrophoresis (2-DE) were developed through the evaluation and modification of existing methods. Among the methods evaluated, the best results were obtained when Tris-HCl, KCl, and phenol extractions were followed by precipitation with organic solvents to purify and concentrate the samples. The utility of the Tris-HCl extraction method was demonstrated on the leaves of six genetically different Citrus varieties. The methods described are versatile and result in high resolution 2-DE gels of Citrus leaf proteins.