In the present study, cyclosporine A (CsA) was successfully incorporated into cationic chitosan nanoparticles by spray-drying method aiming ocular application. Physicochemical characterisation of particles was performed in detail. Among the particles prepared using three types of chitosan with different molecular weights, particles containing chitosan with medium molecular weight was selected for in vivo studies. Selection was dependent on higher incorporation and encapsulation efficiencies of CsA and also better release characteristic in simulated tear fluid. Sheep were used in in vivo studies. Biological samples were collected at predetermined time intervals and were analysed by enzyme immune assay. CsA could be detected in both aqueous and vitreous humour samples for the duration of 72 h. In vivo release profiles indicated prolonged release of active agent from positively charged chitosan formulations. This may be attributed to enhanced residence time at the corneal and conjunctival surfaces.