A rapid and sensitive LC-MS/MS method for the determination of vardenafil and its major metabolite, N-desethylvardenafil, in human plasma using sildenafil as an internal standard was developed and validated. The analytes were extracted from 0.25-mL aliquots of human plasma by liquid-liquid extraction, using 1mL of ethyl acetate. Chromatographic separation was carried on a Luna C(18) column (50mmx2.0mm, 3mum) at 40 degrees C, with an isocratic mobile phase consisting of 10mM ammonium acetate (pH 5.0) and acetonitrile (10:90, v/v), a flow rate of 0.2mL/min, and a total run time of 2min. Detection and quantification were performed using a mass spectrometer in the selected reaction-monitoring mode with positive electrospray ionization at m/z 489.1-->151.2 for vardenafil, m/z 460.9-->151.2 for N-desethylvardenafil, and m/z 475.3-->100.1 for the internal standard (IS), respectively. This assay was linear over a concentration range of 0.5-200ng/mL with a lower limit of quantification of 0.5ng/mL for both vardenafil and N-desethylvardenafil. The coefficient of variation for the assay precision was <13.6%, and the accuracy was >93.1%. This method was successfully applied to a pharmacokinetic study after oral administration of vardenafil 20mg tablet in Korean healthy male volunteers.