Streptococcus suis is a Gram-positive bacterium responsible for major infections in pigs and economic losses in the livestock industry, but also an emerging zoonotic pathogen causing serious diseases in humans. No vaccine is available so far against this microorganism. Conserved surface proteins are among the most promising candidates for new and effective vaccines. Until now, research on this pathogen has focused on swine isolates, but there is a lack of studies to identify and characterize surface proteins from human clinical isolates. In this work, we performed a comparative proteomic analysis of six clinical isolates from human patients, all belonging to the major serotype 2, by “shaving” the live bacterial cells with trypsin, followed by LC-MS/MS analysis. We identified 131 predicted surface proteins and carried out a label-free semi-quantitative analysis of protein abundances within the six strains. Then, we combined our proteomics results with bioinformatic tools to help improving the selection of novel antigens that can enter the pipeline of vaccine candidate testing. Our work is then a complement to the reverse vaccinology concept.