Abstract A fast, simple and selective HPLC method has been developed for the assay of aciclovir, ganciclovir, and penciclovir in human plasma by coupling HPLC with fluorescence detection. 200 μl plasma, with guanosine 5′-monophosphate as an internal standard, was subjected to protein precipitation with a 7% [v/v] aqueous perchloric acid solution. The 40 μl supernatant was injected into a Diamonsil-5 μm C18 column. Aciclovir, ganciclovir, and penciclovir, with solvents composed of methanol and 0.08% aqueous trifluoroacetic acid solution, were analysed by fluorescence detection at 260 nm (excitation) and 380 nm (emission) using a gradient elution program. The calibration curves of all three analytes were linear between 20 and 2000 ng/ml. The mean absolute recoveries of aciclovir, ganciclovir, and penciclovir were 93.91 ± 1.20%, 97.42 ± 0.75%, and 99.01 ± 3.30%, respectively. The mean inter-day CVs for aciclovir, ganciclovir, and penciclovir, were within 1.29–7.30%, 1.00–5.53%, and 1.19–3.54%, respectively. The intra-day bias for aciclovir, ganciclovir, and penciclovir ranged from −2.01 to 6.33%, 1.81 to 7.37%, and 1.42 to 6.91%, respectively. The method has been validated and applied in pharmacokinetic studies in Chinese adult renal transplant patients.