The cytotoxic pattern of cosmetic or pharmaceutical compounds within different layers of the human cornea is of special interest with respect to ocular safety testing. The aim of this study was to evaluate the ability of a newly developed human corneal keratocyte (HCK) cell line as an in vitro model to predict toxicity towards keratocytes in the corneal stroma. The cytotoxic response of immortalised HCK cultures towards different surfactants was compared to that of primary cultures of human corneal keratocytes. Our studies revealed comparable results for immortalised and primary keratocytes. Furthermore, we quantified surfactant-induced cytotoxic effects on immortalised cultures of corneal epithelium and endothelium. In conclusion, the HCK cell line represents an appropriate model to test keratocyte-specific toxicity and may serve as a useful building block in the construction of three-dimensional human cornea equivalent models.