Bioavailability of topical ophthalmic products is substantially determined by the type and concentration of excipients. In addition to the use in conventional ophthalmic formulations, excipients are increasingly utilized in the development of innovative nanosystem-based ophthalmic formulations, in which excipients are supramolecularly organized into nano-sized structures, which can specifically interact with the anterior eye segment barriers and enhance the drug eyerelated bioavailability. Evaluation of the drug transcorneal permeability is a critical step in predicting eye-related bioavailability of topical ophthalmic drugs. In this thesis, the effect of unimers and supramolecular aggregates of the ophthalmic excipients on corneal barrier properties and ophthalmic drug permeability was evaluated, using in vitro (HCE-T cell-based) and/or ex vivo (excised porcine cornea) corneal models. Histological and functional characterization of the in vitro corneal model revealed variability of the HCE-T barrier phenotype, which highlights the important aspects to be considered in the further model development. HCE-T cell-based model of a specific histological structure, but showing improved barrier tightness and a very strong correlation of ophthalmic compounds permeability with excised porcine cornea, was shown to be suitable for the ophthalmic drug permeability evaluation considering the influence of ophthalmic excipients. Biopharmaceutical evaluation of melatonin-loaded nanosystems using HCE-T cell-based models revealed significant influence of chitosan on nanosystem biopharmaceutical properties. In lecithin/chitosan nanoparticles the presence of chitosan ensured mucoadhesive properties and prolonged melatonin release, controlling melatonin in vitro transcorneal permeation, which may provide enhanced melatonin eye-related bioavailability and therapeutic effect. The in vitro/ex vivo evaluation of the effect of nonionic surface active ophthalmic excipients on the ophthalmic drug permeability revealed their concentration-dependent permeability-decreasing effect, which was potentially related to the association of ophthalmic drugs with selfaggregates of surface active ophthalmic excipients. The results of this thesis provide valuable insight into the effect of ophthalmic excipients on ophthalmic drug transcorneal permeability, which may contribute to more directed and economical development of both conventional and innovative topical ophthalmic products.