Immune stimulating complexes (ISCOMs) incorporating recombinant hepatitis B surface antigen (rHBsAg) were prepared for induction of humoral and cellular immunity by subcutaneous administration. Prepared ISCOMs were characterized for their size, shape, incorporation efficiency, zeta potential, antigen integrity, antigen conformation and immunogenicity by biophysical and immunological techniques including transmission electron microscopy (TEM), Dynamic light scattering (DLS), SDS-PAGE, fluorescence spectroscopy, in vitro potency test and in vivo humoral and cellular immune stimulatory efficacy in Balb/c mice. Prepared ISCOM particles show characteristic cage like morphology with average size of 44 ∼nm, polydispersity index 0.1, negative zeta potential (-21.7 mV) and antigen association efficiency - 39%. Tryptophan emission fluorescence and in vitro potency assay data suggest that association of rHBsAg with ISCOMs results in local electrostatic interactions, motional restriction of tryptophan residues of the protein resulting in reduction of anti-rHBsAg monoclonal antibodies binding affinity. Immunization with rHBsAg ISCOMs resulted in upregulation of specific cellular (IFN-γ and IL-2) as well as IgG response (IgG2a isotype biased) humoral response in Balb/c mice. Immune responses were significantly higher than those produced by of alum-adsorbed antigen (alum-rHBsAg) after (one booster) (p < 0.001). These data demonstrate that although the conformation of rHBsAg after incorporation into ISCOMs was moderately altered but due to strong adjuvant ability, rHBsAg ISCOMs were highly immunogenic as compared to marketed rHBsAg formulations by subcutaneous route of administration.