Retinal degeneration is a hallmark of various degenerative ocular diseases, and its accurate quantification is crucial for non-clinical pharmacological studies. Here, we describe a method to assess retinal atrophy by measuring the layers’ thickness in histological sections stained with hematoxylin and eosin. We use a semi-supervised approach with topological penalization to segment layers followed by thickness measurements along the layers’ medial axes. The method is tested on mice for four layers, obtaining a high correlation with manual measurements collected by biologists.