To identify the exact spot position of human, rat and chicken ribosomal proteins (RP) separated by two-dimensional polyacrylamide gel electrophoresis (2-DE), a 2-DE system was designed to separate RP with a pI>8.6 according to their charge in the first dimension and to their molecular mass in the second dimension. Individual proteins were excised from the gels and identified by mass spectrometry after digestion by trypsin. In addition, a mixture of purified RP from these three species was also analyzed by tandem mass tag spectrometry. By combining those two methods 74 RP from human, 76 from rat and 67 from chicken were identified according to the nomenclature initially defined for rat liver RP and by using the Swiss-Prot/trEMBL databases. Whereas human and rat RP were well described, most of RP from chicken were not characterized in databases, since 35 out of 67 chicken RP identified in this study were not listed yet. We propose here the first comprehensive description of chicken RP and their comparison to those from human and rat.