Abstract: A RP-HPLC method was developed for the determination of aeyclovir (ACV) in human plasma. ACV was extracted from plasma using ODS, obtained from Sep-Pak C₁₈ cartridge (Waters associates) and reinstalled in a self-made glass tube suitable for extraction of solutes from small volume plasma samples. After extraction, and rinsing the ODS extract with water to remove hydrophilic impurities, ACV was recovered with methanol and chromatographed on YWG C₁₈H₃₇ column, and detected at 254 mm. The mobile phase was 5.0% (v/v) methanol/water. The average recovery of ACV was 73. 5%; the minimum detection concentration of ACV in plasma was 20 ng/ml with a plasma volume of 0. 2 ml and S/N value of 2. A good linear relationship between the peak area ratios and ACV concentrations was found at the ACV concentrations ranging from 0. 2 to 1.0 μg/ml, C_ACV=0. 840 A_ACV/A_is+0.002, r=0.9969.