A method for the determination of digoxin in human serum using a liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) technique is reported. Digoxin and the internal standard, [21,21,22-(2)H(3)]digoxin, were extracted from 250 mul of human serum using a solid phase extraction cartridge (Oasis HLB) and analyzed by LC/ESI-MS/MS in the selected reaction monitoring mode. The intra- and inter-assay reproducibility and accuracy were satisfactory within the quantification range of 0.20-3.20 ng/ml. The concentrations of digoxin in the serum samples obtained from digitalized patients (n=19) were in the range of 0.25-2.84 ng/ml, which were compared to those obtained by radioimmunoassay.