An HPLC-UV method was developed and validated for the determination of acyclovir in vitreous humor. The method was carried out in isocratic mode using 0.02 mol/L acetic acid/methanol (95:5) as mobile phase, a C18 column at 25 degrees C and UV detection at 254 nm. The method was linear (r2> 0.99) over the range of 35-700 microg/mL, precise (RSD <5%), accurate (recovery ranged from 98.18 to 99.64%), robust, selective regarding of the vitreous humor, and robust remaining unaffected by deliberate variations in relevant parameters. The validated HPLC-UV method can be successfully applied to determine acyclovir directly injected into the vitreous cavity of rabbits' eye.