Purpose: To evaluate methotrexate penetration and distribution profile in ocular structures after short low current transscleral hydrogel iontophoresis. Methods: Methotrexate iontophoresis was studied in rabbits using drug-loaded hydrogels mounted on a portable iontophoretic device. Drug distribution profile was evaluated 2, 4, and 8 hours after iontophoretic treatment of 1.6 mA/cm2 for 4 min. Ocular drug levels were also determined two hours after iontophoretic treatment of 5 mA/cm2, compared to mock iontophoresis and intravitreal injection of methotrexate. Results: Therapeutic drug levels were maintained for at least 8 h at the sclera and retina and for 2 h at the aqueous humor following the iontophoretic treatment. After increasing the current density, a twice-higher concentration was achieved at the vitreous and 8 to 20 time higher concentrations at the retina and sclera.Conclusions: A short low current non-invasive iontophoretic treatment using methotrexate-loaded hydrogels has a potential clinical value in treating ocular inflammatory diseases and intraocular lymphoma.