Mass administration of vaccines against particular disease to produce protective immunity is the ultimate goal of developing recombinant vaccine antigens. Preclinical optimization and standardization of antigen dose is highly crucial for the clinical development of vaccines. In this present study, we have optimized the dose of HBsAg, Dengue and JEV recombinant antigens, through estimation of antibody titer by ELISA method (IgG, IgG1 and IgG2a) and Flow cytometric immunophenotyping of CD4 + and CD8 + surface markers in vivo in BALB/c mice and determined the minimum detectable antigen dose for immunization as well as antibody reactivity.