Aim: Treatment of exudative age-related macular degeneration by using vascular endothelial growth factor (VEGF) antagonists is the gold standard today. So far, several bioactive molecules have been approved for therapeutic use. In this study, we investigate the effects of ranibizumab (Lucentis®), bevacizumab (Avastin®), and aflibercept (Eylea®) on primary human retinal pigment epithelial (hRPE) cells in vitro. Methods: hRPE cells were prepared from donor eyes and cultured under standard culture conditions. Scleral fibroblasts also prepared from donor tissue served as physiological controls. The impact of the anti-VEGF molecules on cell viability was investigated with the trypan blue exclusion assay, whereas proliferation was measured using the MTT assay. Biological activity of the molecules was quantified in a VEGF-enzyme-linked immunosorbent assay (ELISA). Results: All tested substances were biologically active in vitro. They displayed no cytotoxicity on RPE cells or scleral fibroblasts. However, proliferation of RPE cells was significantly decreased after treatment with ranibizumab or bevacizumab but not with aflibercept. Conclusions: The humanized antibodies (fragments) interfered specifically with the RPE cells. The thereby measured inhibition of cell proliferation may indicate possible side effects on the physiology of RPE cells.