To investigate the pharmacokinetics of adefovir as an anti-hepatitis B virus drug, a sensitive and specific liquid chromatography/tandem mass spectrometry method was developed and validated. After a simple protein precipitation using methanol, the post-treatment samples were analyzed on a C(18) column interfaced with a triple-quadrupole tandem mass spectrometer using positive electrospray ionization. A structural analogue, PMPA, was used as the internal standard. The method was linear in the concentration range 0.25-100 ng/mL. The lower limit of quantification was 0.25 ng/mL. The intra- and inter-day relative standard deviation over the entire concentration range was 5.7% or less. The accuracy determined at three concentrations (0.75, 10 and 80 ng/mL for adefovir) was within +/-2.5% relative error. The method described here was successfully applied for the evaluation of pharmacokinetic profiles of adefovir after single oral administration doses of 5, 10 and 20 mg adefovir dipivoxil to ten healthy volunteers.