To evaluate the pharmacokinetics of digoxin in humans, a sensitive and specific LC/MS/MS method was developed and validated for the determination of digoxin concentrations in human plasma. The method was shown to be more sensitive, specific, accurate, and reproducible than common techniques such as RIA. For detection, a LC/MS/MS system with electro spray ionization tandem mass spectrometry in the positive ion-multiple reaction-monitoring (MRM) mode was used to monitor precursor to product ions of m/z 798.5–51.5 for digoxin and m/z 782.5–35.5 for the internal standard, digitoxin. The method was validated over a concentration range of 0.02–5ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was above 80%. Imidafenacin, coadministered in a drug–drug interaction study, had no detectable influence on the determination of digoxin in human plasma. The novel method was applied to a drug–drug interaction study of digoxin and imidafenacin and the characterization of steady-state pharmacokinetics of digoxin in humans after oral administration at a dose of 0.25mg on days 1 and 2 followed by 0.125mg daily doses on days 3 through 8.