OBJECTIVE The purpose of this study was to establish a reliable and reproducible method to isolate and cultivate canine corneal epithelial and stromal cells (cCECs and cCSCs). The cells were subsequently used for in vitro testing of topically applied diluted povidone iodine (PVI). METHODS Corneas of dogs, euthanized for reasons unrelated to this study, were used to collect primary cCECs and cCSCs. Corneas were enzymatically digested and explants obtained using biopsy punches. Epithelial and stromal explants were separately taken into culture. Cell proliferation and migration was evaluated after incubation of cCECs and cCSCs with PVI in different concentrations (1, 2, or 5%) and with different exposure times (1, 3, or 10 min). RESULTS Solely incubation of 4 mm diameter corneal epithelial explants in a 48-well culture plate in full medium led to sufficient growth of cCECs. Up to four passages were achieved with a cell density of 10,000-20,000 cells/cm2 after dissociation of cells in trypsin for 8 min. Cell detachment and passaging for cCSCs were possible with almost every cornea and explant. Canine CSCs were less sensitive to PVI in all concentrations and over time than cCECs. Epithelial and stromal cell proliferation and migration decreased with increasing exposure times and PVI concentrations across all groups. CONCLUSIONS The described method is a straightforward and sound way to isolate and cultivate cCECs and cCSCs in vitro. Basic information on cCEC and cCSC migration and proliferation after incubation with PVI, was gathered. The results may provide a step towards an optimal preoperative antisepsis protocol for ophthalmic surgery in future.