To assess how safe and effective it is to use menthol as a permeability enhancer in ophthalmic drug delivery systems. In this study, the effect of menthol on permeability of dexamethasone disodium phosphate in the cornea and sclera was investigated in vitro. Application of topical drops and subconjunctival injection of dexamethasone disodium phosphate with or without 0.1% menthol was administered to rabbit eyes, and the drug concentration was detected in aqueous humor, cornea, vitreous, and retinochoroidal tissues. The safety of menthol was assessed on the basis of corneal hydration level, Draize test, electroretinography (ERG), and histological examination. 0.05% and 0.1% menthol significantly enhanced the penetration of dexamethasone in the cornea, but did not change the dexamethasone penetration in sclera in vitro. When topical drops of dexamethasone containing 0.1% menthol were administered, a significantly increased concentration of dexamethasone in the cornea and aqueous humor tissues was reported, but dexamethasone concentrations remained unaffected in the retina–choroid tissues. On the other hand, increased drug concentration in aqueous humor, cornea, vitreous and retinochoroidal tissues was achieved through subconjunctival injection. No signs of irritation were observed when menthol was administered at concentrations ranging from 0.025%-0.1%; moreover, no substantial toxic reactions were observed in corneal hydration level, electrophysiological, or histological examinations after the addition of 0.1% menthol. Menthol may improve the ocular penetration of a drug in a transcorneal and transscleral drug delivery system without causing toxic reactions.