Purpose: The model drug norfloxacin (NOR) was encapsulated into trehalose (TRH) and hydroxyethylcellulose (NAT) microspheres to obtain a novel gelling ophthalmic delivery sys-tem for prolonged release on corneal tissue. Methods: We assessed NOR release from microspheres, prepared by the emulsion-solvent evaporation method. A new in vitro tear turnover model, including inserts containing recon-stituted human corneal epithelium (RHC), was designed to evaluate the TRH/NAT micros-pheres’ precorneal retention time. Bioadhesive properties of TRH/NAT microspheres were validated by using drug-loaded microspheres prepared with gelatine (GLT) commonly used as reference material in adhesion studies. Results: In vitro drug release showed a typical trend of swelling systems. Precorneal re-tention tests showed that TRH/NAT microspheres maintained fluorescence in tear fluid for 81.7 min, whereas TRH/GLT microspheres and water solution maintained fluorescence for 51.8 and 22.3 min, respectively. NOR released from microspheres permeated throughout RHC slower (Js = 23.08 µg/cm2h) than NOR from commercial eye drops (Js = 42.77 µg/cm2h) used as the control. Conclusions: Adequate drug concentrations in aqueous humor could be prolonged after the administration of TRH/NAT/NOR microspheres. Good bioadhesive properties of the system and slow drug release on corneal surface might increase ocular NOR bioavailability.