The human milk is considered the best nutrient to the human newborn. Its quality is not only a function of its nutritional values but also of the presence of bioactive compounds which facilitate the transition of the life of its intrauterine state for the extra-uterine state. Although several of these substances have been identified, many still wait for their identification, mainly the less abundant proteins. The aim of the present work was to develop a methodology for comparing the proteome of the colostrum and mature milk wheys by using two-dimensional electrophoresis and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry. Samples of colostrum (within 1 week postpartum) and mature milk (1-3months postpartum) were collected in the Hospital Universitário Pedro Ernesto (Rio de Janeiro- Brazil), after agreement of the women. Samples of colostrum and mature milk were acidified with 1N acetic acid and centrifuged at 16.500 x g for 30 minutes at 4ºC. The wheys were treated with Cibacron Blue 3GA-Agarose (Sigma) for adsorption of the most abundant proteins (a- lactalbumin, lactoferrin and immunoglobulin A), in order to improve the visualization of the less abundant proteins, which correspond to the target of this study. An efficient removal of the major proteins of colostrum and mature milk samples were obtained when proteins adsorbed to Cibacron Blue 3GA-agarose were eluted with 0.02mM sodium phosphate buffer, pH 7.2, containing 0.5 mM NaCl. The eluted fraction, enriched in the less abundant whey proteins of both samples (colostrum and mature milk), were treated with the Clean up kit (Amersham Bioscience do Brasil Ltda) and then analyzed by two-dimensional electrophoresis using the MultiphorTM II System (Amersham Biosciences). The proteins were fractionated by isoelectric focusing (IEF) in immobilized gradient of pH (3-10) in the first dimension, followed by gradient SDS PAGE (8 to 18% in respect to acrylamide monomers) in the second dimension. The gels were stained with Coomassie Blue G-250. The visualized spots were cut, digested with trypsin and will be identified by MALDI-TOF Mass spectrometry.