The in vivo behaviour of 5% gentamicin sulphate ocular mini-tablets (2-mm diameter, 6.525 mg weight) was compared with gentamicin eye drops in six ponies. Two mini-tablets were inserted on the bulbar conjunctiva of the right eye while a similar dose of gentamicin was administered via eye drops in the left eye. Irritation induced by the mini-tablets and the eye drops was evaluated using a visual analogue scale (0-10). Tears were sampled with ophthalmologic absorption triangles for 1 min for the determination of the concentration of gentamicin sulphate using a microbiological plate diffusion method. Irritation induced by the tablets was minor and clinically acceptable (overall median score of 1.7 ± 1.4). Eye drops induced a sharp increase in gentamicin sulphate concentration (364.4 μg/mL after 5 min) followed by a fast decline (10.8 μg/mL after 60 min). The increase in concentration induced by the ocular mini-tablets was less pronounced (up to 56.2 μg/mL after 30 min) and followed by a gradual decrease; the concentration remained above 15 μg/mL for 8 h. Ocular 5% gentamicin sulphate mini-tablets are clinically well-tolerated in ponies, assuring a constant concentration in the tears for at least 8 h.