Proteome analysis is becoming a powerful tool in the functional characterization of organisms, and takes a broad, comprehensive, systematic approach to understanding biology. Following the sequencing of the phytoplasma genomes, the next step is to characterize the expressed proteome of phytoplasmas to acquire the verification and functional annotation of all predicted genes and their protein products. Here, we describe the protocol of mulberry dwarf phytoplasma purification, phytoplasma protein extraction and separation by SDS-PAGE, in-gel tryptic digestion of the proteins, separation of the digested peptides by liquid chromatography, and identification of the peptides by mass spectrometry. The protocol described here is also applicable to the analysis of other phytoplasma proteomes.