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Summary: Organs-on-chips are microfluidic devices for cell culturing to simulate tissue- or organ-level physiology, providing new solutions other than traditional animal tests. Here, we describe a microfluidic platform consisting of human corneal cells and compartmentalizing channels to achieve fully integrated human cornea’s barrier effects on the chip. We detail steps to verify the barrier effects and physiological phenotypes of microengineered human cornea. Then, we use the platform to evaluate the corneal epithelial wound repair process.For complete details on the use and execution of this protocol, please refer to Yu et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.