Abstract The utility of both in vitro and in vivo methods to assess the release pattern of alginate, HPMC (TTAL) composite microspheres containing stabilized tetanus toxoid (TT) vaccine were evaluated. TT was stabilized with different concentrations of stabilizer, lactose (1–5%, w/v) and encapsulated into alginate, HPMC composite microspheres by W/O/W multiple emulsion method. The morphology of the prepared microspheres was smooth and spherical in shape with a diameter of around 10μm. The in vitro release efficiency of microspheres was evaluated for the period of 70 days. In TTAL microspheres, 4% (w/v) lactose gave good sustained antigen delivery for the period of 70 days. Antigen releases from microspheres were determined by ELISA. Based on the results of in vitro release, the ideal batch of TTAL was used to carry out the in vivo studies by antibody induction method using alum adsorbed tetanus toxoid (CRIT) as standard. The antibody level was measured for about of 9 months and finally with one booster dose after 12 months. In this case, TTAL antibody level was rose up to 3IU/ml of guinea pig serum and 2.5IU/ml of guinea pig serum was observed in CRIT after 1 year with second booster dose. This novel approach would be helpful to replace the existing adjuvant alum in future.