There is reported clinical evidence of retinal pigment epithelium (RPE) alterations after intra-arterial (IAC) and intravitreal chemotherapy (IViC) of retinoblastoma. The purpose of this study was to investigate the cellular toxic eﬀects of melphalan, topotecan and carboplatin on the RPE in a cell culture model. The eﬀects of melphalan, carboplatin and topotecan on ARPE19 cell morphology were examined by phase contrast microscopy. Cell proliferation was quantiﬁed by BrdU incorporation, cell viability studied via MTS assays, and cell densities were estimated by Crystal Violet staining, and apoptosis induction studied via caspase 3/7-activity assays after a 24-hour incubation period. Staurosporine, media without fetal bovine serum, diluents of melphalan, carboplatin and topotecan were applied as positive and negative controls, respectively. A concentration-dependent increase in the number and size of gaps in the ARPE19 cell layer with each drug was observed. There was a signiﬁcant decrease in proliferative activity and cell viability of RPE cells as well as an increase in apoptosis after 24 hours culture in media supplemented with melphalan and topotecan. Carboplatin had comparable eﬀects on cell proliferation and cell viability; however, no signiﬁcant apoptotic impacts were observed. The three cytostatic drugs had insigniﬁcant eﬀects on cell density measurements. Morphological monitoring and toxicity assays indicate a direct toxic eﬀect of melphalan and the other two cytostatic drugs on ARPE19 cells. Thus, a direct toxic eﬀect of melphalan in vivo after IAC or IViC on the RPE seems probable and may explain the clinical and angiographic RPE alterations observed in some retinoblastoma patients.