Unsatisfactory performance of the existing BCG vaccines, especially against the adult pulmonary disease, has urged the need for an effective vaccine against tuberculosis (TB). In this study, we employed differential proteomics to obtain a list of antigens as potential vaccine candidates. Bacterial epitopes being presented at early stages on MHC class I and class II molecules of macrophages infected with Mycobacterium tuberculosis (M. tb) were identified using iTRAQ labelling and reverse phase LC-MS/MS. The putative vaccine candidates, thus identified, were tested as plasmid DNA vaccines in mice to ascertain their protective efficacy against the aerosolized M. tb challenge, based on their ability to reduce the bacterial load in the lungs of infected mice. Here, we observed that 4 out of the 17 selected antigens imparted significant protection against the challenge of M. tb. The four shortlisted antigens were further assessed in a more stringent guinea pig model, where too, they demonstrated.significant protection. It concludes that combining a proteomics approach with the in vivo assessment of vaccine candidates in animal models can be valuable in identifying new potential candidates to expand the antigenic repertoire for novel vaccines against TB.