In this study, genomics- and proteomics-based approaches were used for the identification of Streptococcus pneumoniae potential biomarkers for diagnostics and prospective targets for treatment. Through a Pan-genome analyses 11 strains from the CCUG demonstrating genetic variability within the species were selected for a proteomic characterization. Mass spectrometry-based proteomics in combination with bacterial surface shaving were used to study the surface proteome of S. pneumoniae. The 11 strains were analyzed in triplicates by proteotyping (typing and characterization of species by mass spectrometry-based proteomics) and bioinformatic approaches, including the Microorganism Identification and Classification, were used to validate the expressed proteins and peptide biomarkers.