SummaryHigh concentrations of digoxin-like immunoreactive substances (DLIS) artificially increase serum digoxin concentrations. However, DLIS are absent in the protein-free ultrafiltrate because of their strong binding with serum macromolecules, whereas ≈75% of digoxin can be found in the ultrafiltrate. Using regression analysis, we devised equations by which total digoxin concentration can be calculated from free digoxin and albumin concentrations in serum. We used two different assays, fluorescence polarization and chemiluminescence, for measuring total and free-digoxin concentrations in sera. Both equations were very similar. Because measured concentrations of digoxin in the serum exhibit DLIS interferences, the measured concentrations were sometimes higher in volume-expanded patients than the calculated digoxin concentrations. We also estimated the extent of interferences from DLIS by subtracting the calculated digoxin concentration from the measured digoxin concentration in volume-expanded patients.