The aim of our study was a systematic analysis of the impact of variable parameters on indocyanine green (ICG) and trypan blue (TB) related cytotoxicity on human RPE cells. ARPE-19 cells were incubated with ICG (5.0-0.025mg/ml), with ICG-free solutions of corresponding osmolarities or with TB (1.5-0.0375mg/ml). Incubation lasted 1-20min with or without endolight illumination for 1min or 5min. Cell viability and morphology were examined after 6h, 24h and 72h to detect acute and delayed effects. In the absence of endolight, ICG cytotoxicity depends on osmolarity and exposure time. In the presence of endolight, cytotoxic effects are influenced by dye concentration. TB cytotoxicity depends on dye concentration and exposure time, but not on illumination. All observed cytotoxic effects were mainly acute. Both ICG and TB can be cytotoxic depending on concentration and exposure time. ICG related cytotoxic effects are additionally determined by osmolarity and phototoxicity. However, concentrations (<1mg/ml) and incubation times (<5min) as used in clinical practice would appear to be well tolerated.