Chitosan adsorbed microspheres containing tetanus toxoid were prepared in the size range of 10 μm to 75 μm, by emulsion-cross linking technique at different speeds of agitation. The amount of tetanus toxoid incorporated into chitosan microspheres were estimated by limes flocculation test and in vivo evaluation of tetanus toxoid adsorbed chitosan microspheres were determined by toxin neutralization method using albino mice. The results of in vivo release for the batches of 10 μm and 25 μm correlates with the results of in vitro in which both the batches passes the limit of IP standard (4 Lf) where as, for the batches of 50 μm and 75 μm, the in vitro release of tetanus toxoid was 2 Lf. But our in vivo studies for the batches of 50 μm and 75 μm fail to pass the limit stated in IP. The release of tetanus toxoid from the chitosan microspheres was found to be sustained for the period of 6 months.