The biocompatibility of materials used in biomedical applications, especially those in direct contact with human tissue, is crucial to ensuring their safety. Ensuring material biocompatibility requires a wide range of in vitro and in vivo tests, with in vitro tests using cell culture systems being the first step in biomaterial characterization. Among the commonly used methods for assessing cell viability are colorimetric tests, such as MTT and LDH assays. While these assays provide valuable information about cell viability, their results can be affected by biochemical substances. This study focused on evaluating the reliability of MTT and LDH assays in nicotinamide-supplemented medium, which optimized culture conditions for the differentiation of ARPE-19 cells. The results were compared with a live/dead viability test based on fluorescence staining, providing insight into the effectiveness of different cell viability assessment methods in this specific context. This research is important in developing biomaterials for retinal prostheses, where maintaining high biocompatibility is essential for successful implantation.