Objective: Acute corneal permeability change after instillation of benzalkonium chloride (BAC) was evaluated using a newly developed in vivo corneal transepithelial electric resistance (TER) measurement method. Method: Corneal TER was measured by Ag/AgCl electrodes placed in the anterior aqueous chamber and on the cornea of live rabbit eyes. TER was measured and TER change after instillation of 0.05% BAC solution was monitored. After TER measurement, cornea was excised and fixed for transmission and scanning electron microscopy. For the control study, physiologic saline was used instead of BAC. Results: The TER of normal rabbit cornea was 602.3 ± 195.0 Ωcm<sup>2</sup>. TER decreased instantly after instillation of 0.05% BAC. In 5 s, TER decreased to 58.3 ± 5.2%. In 60 s, TER decreased to 18.5 ± 3.2%. At all time points, TER after instillation of 0.05% BAC was significantly lower than that of the control (p < 0.0001). Dissociation of tight junctions and the destruction of superficial cell membranes were observed under electron microscopy. Conclusion: Corneal epithelial change with increased permeability is rapid and intense after the instillation of highly concentrated BAC solution, accompanied by disorder of tight junctions and cell membranes of superficial cells. The newly developed in vivo corneal TER measurement method is suitable for assessing acute corneal change after drug instillation.