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Ocular iontophoresis has been investigated for many years as a non-invasive technique for enhancing ionized drug penetration through ocular tissues. In this study we assessed the penetration of charged fluorescent nanoparticles into rabbit eyes using hydrogel iontophoresis. Particle distribution into ocular tissues and penetration efficiency of negative nanoparticles compared with positive nanoparticles was also evaluated. Cathodal and anodal iontophoretic administrations were performed using polyacrylic hydrogels loaded with charged nanoparticle suspension (20–45 nm), applying a current intensity of 1.5 mA for 5 min onto the cornea and sclera. At pre-set time points post treatment, eyes were dissected and tissues were evaluated for fluorescence intensity. Strong fluorescence evidence was observed at anterior and posterior ocular tissues. Negative particle distribution profile revealed fast uptake into the outer ocular tissues, within 30 min post treatment, followed by particle migration into the inner tissues up to 12 h post treatment. The positively charged particles demonstrated better penetration abilities into inner ocular tissues compared to the negatively charge particles. This work provides an opening for the development of a new ocular therapeutic pathway using iontophoresis of extended release drug-loaded charged nanoparticles.