To assess the usefulness of noninvasive combined technologies used to observe ocular surface changes in toxicology studies. Benzalkonium chloride (BAC) at 0.01%, 0.1%, 0.25%, and 0.5% was applied to rat corneas for 11 days. The eye was evaluated macroscopically from day (D)0 to D52. The cornea was examined with the slit lamp, a fluorescein test was performed, and a confocal microscope was used in vivo to calculate corneal thickness, score corneal epithelial and endothelial defects, and quantify corneal stromal inflammation and neovascularization. Conjunctival impression and brush cytology specimens were taken for labeling with MUC-5AC antibodies and sub-G1 peak analysis by flow cytometry, respectively. Histologic analyses were performed on D11. Although macroscopic and slit lamp examinations revealed signs of ocular irritation in the 0.25% and 0.5% BAC-treated eyes only, in vivo confocal microscopy revealed epithelial defects in the 0.01% and 0.1% BAC-treated corneas, and sub-G1 peak analyses showed increased apoptosis for all the BAC concentrations on D8 and D11. BAC at 0.25% and 0.5% induced increased corneal thickness, loss of goblet cells, reversible corneal inflammation, and persistent neovascularization. Sub-G1 peak analysis of conjunctival brushings, in conjunction with in vivo confocal microscopy of the cornea and immunolabeling of conjunctival imprints, constitutes a noninvasive, reliable, and sensitive tool to evaluate toxic drug-induced ocular surface damage in rats, in addition to standard clinical assessments and at a wide range of concentrations, including the lowest ones. This study is consistent with the international strategy aimed at reducing the use of animals and refining animal toxicologic models.