Introduction: The influence of photorefractive keratectomy (PRK) on drug penetration into the eye is not yet established, as this procedure may alter the barrier function of the cornea as well as the blood ocular barrier. Pupillary response to topical tropicamide depends on its penetration into the anterior chamber. The purpose of this study was to examine the influence of PRK on pupillary response to topical tropicamide, and hence to evaluate whether PRK alters drug penetration into the eye. Methods: Pupillary diameter was measured using infrared pupillometer at 0, 15 and 30 min after instillation of 0.1% tropicamide to both eyes of 28 patients 4 weeks after PRK in one eye. The untreated eye served as control. Results: Pupillary diameter before and 30 min after dilation was 7.4 ± 1.2 and 8.0 ± 1.0 mm in the treated eyes and 7.5 ± 1.2 and 8.1 ± 1.0 mm in the untreated eyes, respectively (p = 0.45). Percentage of pupillary dilation (Δ pupillary diameter/pupillary diameter at 0 min ×100) was also similar in treated and untreated eyes for the entire study group (12.5 and 10.5%, respectively, p = 0.17). A trend for greater dilation was seen in patients with myopia above 6 diopters (13.7 and 10.5% in treated and untreated eyes, respectively, p = 0.11) and cornea thinner than 540 µm (14.2 and 10.1% in treated and untreated eyes, respectively, p = 0.1). Discussion: Our results confirm animal and human studies that found restoration of the ocular drug barrier at 4 weeks following PRK. The trend for greater penetration in patients with high myopia and thin corneas warrants further study on a larger cohort of this subgroup.