Chromovitrectomy represents a recent development in the field of vitreoretinal surgery. Several dyes are available for intraocular application with different staining characteristics. Before the intraoperative use in humans, new dyes need to be evaluated thoroughly in experimental in vivo and ex vivo studies in order to detect potential adverse effects related to dye toxicity. This article describes a reasonable approach for the assessment of novel dyes prior to the use in humans.