Excimer laser photorefractive keratectomy (PRK) is a well-established procedure which is frequently applied to correct myopia. Since structural alterations of the corneal epithelium occur after the treatment, a different drug permeation can be assumed. To investigate the effects of PRK on drug permeation, excimer laser ablations with varying depths were performed on isolated pig eyes. The permeation of lipophilic (diclofenac-sodium; D-Na) and hydrophilic (pilocarpine-hydrochloride; P-HCl model drugs were studied in vitro. Under these experimental conditions, P-HCl demonstrated a significant (p < 0.05) enhancement of permeation in relation to the ablation depth. In contrast, corneal epithelial thickness scarcely influenced the permeation rate of D-Na. Not until removing the entire epithelium did a significantly increased permeability occur, when compared to untreated cornea. These results suggest that PRK may significantly reduce the corneal barrier function and alter pharmacokinetics of topical medication.