Acyclovir and its major impurity guanine are determined rapidly by the incorporation of a monolithic column (100 mm x 4.6 mm i.d., Merck) to an automated HPLC system. A simple flow gradient protocol was adopted in order to accelerate the separation-detection cycle. Using 0.2% CH(3)COOH (pH 3.1) as the mobile phase and detection at 254 nm, guanine was effectively separated from the system peak (t(R)=1.25 min), while the retention time of acyclovir was 2.35 min. Linearity of the assay was validated in the range 0.1-1.0% guanine and 80-120% acyclovir (n=5). The accuracy and within- and day-to-day precision of the method were also validated, while the limits of detection and quantitation of both analytes were determined. The proposed method was successfully applied to the quality control of acyclovir raw material and the quality and stability control of acyclovir-containing pharmaceutical creams (Hagevir 5%, w/w, Cosmopharm Ltd., Korinthos, Greece).