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Purpose: To determine whether excimer laser ablation of guttae is a viable strategy for removal of diseased tissue in Fuchs' endothelial corneal dystrophy (FECD) on excised human Descemet membranes and whether an excimer laser‐created wound on healthy human corneas ex vivo is recolonized with corneal endothelial cells. Methods: Descemet membranes of FECD patients and corneal endothelium of normal human corneas were ablated ex vivo using an excimer laser licensed for glaucoma surgery. Specimens were kept in cell culture medium supplemented with 10 μm of rho‐kinase inhibitor ripasudil. Corneal endothelial cell regeneration was observed using light and electron scanning microscopy. Furthermore, the whole corneal samples were evaluated by haematoxylin/eosin staining and immunohistochemical analysis using antibodies against Na+/K+‐ATPase. Results: Guttae and corneal endothelium could be ablated with an excimer laser without total ultrastructural damage to the Descemet membrane or stroma. Nearly complete endothelial wound closure was accomplished after 26–38 days in treated corneas. Light and electron scanning microscopy suggested the establishment of a layer of flat endothelial cells. Additionally, Na+/K+‐ATPase expression could only be observed on the inner side of the Descemet membrane. Conclusion: Our proof of concept study demonstrated that excimer lasers can be used to ablate diseased tissue from excised FECD Descemet membranes ex vivo. Additionally, corneal endothelial cells recolonize a previously ablated endothelial area in healthy human corneas ex vivo under treatment with ripasudil. Thus, our results are the first experimental basis to further investigate the feasibility of an excimer laser ablation as a graftless FECD treatment option. [ABSTRACT FROM AUTHOR]