Ray-finned fishes represent the most diverse vertebrate lineage and show extensive variations in physiology, ways of life and adaptations to marine and freshwater environments. Actinopterygii are largely exploited for human consumption and several species have been established as biological models. The in vitro culture of cells is fundamental for several fields of biological research, being an alternative for studies that use animals, since it mimics the cellular homogeneity of the original organisms beyond the advantage of reducing the variability of responses observed under in vivo conditions. In the case of fish cell cultures, besides being an important biomedical tool, they can offer important contributions to aquaculture and fish conservation. Hundreds of fish cell lines have been established using specific methods for each cell type and species. Here we describe an optimized protocol for obtaining cell cultures from the caudal fin of a wide range of ray-finned fishes including marine and freshwater species. The proliferative potential of these cultures makes them useful for several applications and the minimally invasive feature of this protocol makes it suitable for use in conservation plans.