Proteomic analyses have emerged in various areas of plant biology. 2-DE (two-dimensional gel electrophoresis) is one of the most efficient and powerful methods to study complex patterns of gene expression at the level of protein. Nevertheless, the major constraint of 2-D protein separation is the quality of the protein extracts to be assayed. The electrophoretic separation of protein from plant tissue extracts is often complicated by other nonprotein contaminants endogenous to the plant, such as organic acids, lipids, polyphenols, pigments, terpenes, etc. High quality whole cell protein extracts from woody plants, especially coffee, is difficult to obtain due to their high amounts of non-protein contaminants. Coffee root tissue is notoriously recalcitrant to common protein extraction methods due to high level of interfering compounds and little amount of protein of the tissue. The objective of this work was to develop a protein extraction protocol for coffee roots to ensure a high-resolution two-dimensional gel electrophoresis for proteomic profiling. Initially, three protocols were individually evaluated and the integrity of the proteins was monitored by SDS-PAGE. The best protein profile was obtained with a combination of these three protocols. Briefly, total protein was extracted from an acetone dry powder that was prepared by several acetone washings of the sample, followed by phenol extraction and precipitation with ammonium acetate in methanol. The efficiency of this combined protocol was monitored by two-dimensional gel electrophoresis. For the first dimension, 100 μg of protein were loaded on isoelectric strip gels in which the pH gradient was established with pH 4-7 ampholines (Multiphor II system). The second dimension was carried out on 12% SDS-polyacrylamide gels. The proteins were visualized by silver staining and colloidal coomassie blue staining. Both staining methods were effective in revealing smear-free spots. The high-resolution two-dimensional gel electrophoresis of coffee root proteins could be related to the efficiency of the modified protein extraction protocol. Although the silver staining was more sensitive and revealed a greater number of spots, the colloidal coomassie blue staining showed satisfactory results with the advantage to allow further MS analyses. Supported by: CAPES and Consórcio Brasileiro de Pesquisa do Café.