Limbal melanocytes (LMs) are found in the corneoscleral limbus basal epithelial layer and interact with neighboring limbal epithelial progenitor cells. The difficulty of isolating and cultivating LMs is due to the small fraction of LMs in the overall limbal population and the frequent contamination of primary cultures by other cell types. This has limited the research on freshly isolated LMs and the investigation of their biological significance in the maintenance of the limbal stem cell niche. Here, we describe an optimized protocol for the efficient isolation and expansion of LMs from cadaveric corneal limbal tissue using CD90 and CD117 as selective markers in fluorescence-activated cell sorting to obtain a pure population of LMs (CD90−CD117+) with self-renewal capacity and sustained melanin production. The isolation of pure LMs from a single preparation enables direct transcriptomic and proteomic analyses, as well as functional studies on freshly isolated LMs, which can be considered the proper counterparts of LMs in vivo and have potential applications in tissue engineering.