Background: The aim of this study is to evaluate the safety profile of Brilliant Blue G (BBG) with and without exposure to light (L) on three different retinal cell lines. Method: ARPE-19, R28 and MIO-M1 cells were treated with BBG: 0.125mg/mL (0.5x clinical concentration), 0.25mg/mL (1x) or 0.5mg/mL (2x) with or without surgical illumination of halogen light exposure for 10 min, 15 min or 30min. Cells were further cultured after 24h and then analysed for cell viability, late stages of apoptosis and mitochondrial damage associated with early apoptosis using assays that measure trypan blue dye exclusion, increases in caspase-3/7 activity or changes in mitochondrial membrane potential (ΔΨm), respectively. Result: All three cell lines that were exposed to BBG in the presence or absence of light exposure for 30min were found to have cell viability and caspase-3/7 activity levels similar to the untreated cultures. The mitochondrial membrane potential (ΔΨm) was decreased significantly at the 2x+L dose and 2x dose in all three retinal cell lines compared to their respective untreated control cells. At the lower doses of BBG, with or without exposure to light, the ΔΨm values were similar to the untreated control cultures. Conclusion: Exposure to BBG dye concentrations that are used clinically (0.125mg/mL and 0.25mg/mL) in the presence up to 30min of surgically equivalent light intensity is safe for retinal cells.