Abstract Purpose Retinal Pigment Epithelial (RPE) cells perform critical functions in the visual cycle. Their melanin pigmentation, which is organized into specialized compartments – melanosomes, is highly critical for proper vision. A chemical method to induce pigmentation in a non‐pigmented model of ARPE‐19 cells was applied using L‐DOPA as a repurposed drug from the current treatment of Parkinson's disease. Methods L‐DOPA was optimized for its toxic effect on ARPE‐19 cells along with pigmentation development. Gene expression and immunocytochemistry confirmed upregulation of melanogenesis‐related genes and proteins. Melanosomes were characterized by TEM. Results We found 1000 μM L‐DOPA to induce pigmentation of ARPE‐19 cells by Day 3, and achieve full pigmentation by Day 5. By Day 5, L‐DOPA at 1000 μM induced mitochondrial and nuclear DNA damage. However, the gene expression of RPE‐specific markers ( tyrosinase , TYRP1 , CRALBP , PEDF ) was significantly different in L‐DOPA‐treated ARPE‐19 cells compared to non‐treated ones. Positive expression for Tyrosinase enzyme was confirmed by ICC on both Day 3 and Day 5 of L‐DOPA treatment. Transmission electron microscopy showed the de novo melanosome formation with ultrastructural features of various stages of maturity (Stage I to IV), apical‐basal polarity and melanosome localization on the apical side of the L‐DOPA‐treated ARPE‐19 cells. Conclusion Our study showed that L‐DOPA treatment could induce de novo melanosome formation in amelanotic RPEs. We propose a newer approach of developing an ex vivo model for de novo pigmentation of RPE cells with cell‐specific modification and culture condition optimization.