Reverse vaccinology has been very successful in the discovery of vaccine candidates against many pathogenic bacteria by integrating genome and proteome mining. This great achievement was facilitated by the complementarity of the in silico prediction of antigens and the empirical data on protein localization, expression, and immunogenicity obtained through different techniques based on electrophoresis, immunoblotting and mass spectrometry. An iterative process between information provided by DNA sequence analysis and proteomic data has been established leading to precise antigen identification. In this review, we report how the identification of surface and exoproteomes of Gram-positive pathogens have contributed to the selection of vaccine candidates. Moreover, we show how quantitative mass spectrometry is now paving the way for identifying protective antigens that play key roles during infection and represent the most promising vaccine targets.