The biocompatibility of liquid artificial vitreous replacements is generally assessed by performing tests in animal models before their clinical use, whereas in vitro experimentation is seldom carried out due to their physico-chemical characteristics. Since their introduction in vitreoretinal surgery, however, the use of some certified vitreous replacements has been discouraged after clinical trials, because of the occurrence of serious side effects. This observation suggests that the tests currently performed for biocompatibility assessment cannot fully guarantee their safety when they are used in humans. Here we review the available literature on in vitro biocompatibility testing of liquid artificial vitreous replacements and survey our own experience on the subject, obtained by using primary retinal cell cultures, seeded on micro-porous inserts. We suggest that in vitro biocompatibility assessment, conducted before experiments in animal models, could improve the required safety evaluation and decrease the risk of undesired side effects, as well as providing a beneficial reduction of animal experimentation.