Francisella tularensis is a Gram negative facultative intracellular bacterium that causes tularemia in humans. There is no licensed vaccine available against this disease. Membrane proteins have been evaluated as vaccine candidates in many bacterial pathogens because of their important roles in initial infection, intracellular survival, virulence and immune evasion. Control of F. tularensis infection is mostly dependent on cell-mediated immune responses, however, recent studies have shown that humoral immunity also plays an important role. A novel approach has been developed in this study to identify putative T-cell reactive antigens using humanized mice along with bioinformatics tools. Membrane proteins of F. tularensis subsp. novicida were extracted by a non-ionic detergent Triton X-114 and fractionated using liquidphase isoelectric focusing and whole gel elution. A total of 83 proteins were identified from fractions that induced strong cell-mediated immune responses. Using bioinformatics algorithms, such as PSLpred, BLASTP, and HLA-DR4Pred, allowed the identification of eight antigens that are membrane or periplasmic proteins, with low homology to human and mouse proteins, and contain putative HLA-DR4 MHC class II binding epitopes. Recall assays measuring cellular cytokine production in F. tularensis subsp. novicida Δiglc immunized HLA-DR4 transgenic mice confirmed that one of the identified proteins, FopB is a T-cell antigen. Moreover, FopB has been shown to react with human convalescent tularemia sera and is homologous to an outer membrane protein of Coxiella. These observations further validated our immunological and bioinformatics screening methods. The approach described in this study provides a useful tool to identify potential vaccine candidates against tularemia with high relevance to humans.