Amikacin is an aminoglycoside antibiotic that has poor corneal penetration due to its hydrophilic properties. The purpose of this study was to compare and evaluate the penetration of amikacin sulfate into aqueous humor of the rabbit eye when applied by different routes and concentrations, namely 100 or 250 mg/ml topical fortified amikacin eye drops, 100 or 250 mg/ml amikacin-embedded soft contact lenses and 25 mg subconjunctival amikacin injection. One hour after application, amikacin was not detectable in any of the 100 mg/ml concentration groups. High levels of amikacin above the minimum inhibitory concentration for susceptible bacteria were detected when applied subconjunctivally and by 250 mg/ml topical fortified routes. Topical fortified amikacin 250 mg/ml reached the highest value in the aqueous (p < 0.05). Our results point out the poor corneal penetration of amikacin in standard concentrations from the intact rabbit cornea and that subconjunctival injections might provide satisfactory penetration.