An over-the-counter Chinese medicine, Chan Su, is used as a cardiotonic agent. We demonstrated significant digoxin-like immunoreactivity in various organic and aqueous extracts of Chan Su. For example, when a 20-microL aliquot of an aqueous extract of Chan Su powder (1 mg/mL) was added to a 2-mL aliquot of a drug-free serum, the observed digoxin-like immunoreactivity was 2.76 ng/mL (3.53 nmol/L) digoxin equivalent using the fluorescence polarization immunoassay (FPIA). The magnitude of interference was much lower (0.94 ng/mL [1.20 nmol/L]) with the microparticle enzyme immunoassay (MEIA), and no interference was observed with the chemiluminescent assay (CLIA). We also observed a significant positive interference of the extract with the serum digoxin measurement using FPIA. In contrast, we observed a negative interference (falsely lowered digoxin concentration) of the extract in the serum digoxin measurement with the MEIA. The extract had no effect on the serum digoxin measurement with the CLIA. By taking advantage of the high protein binding of Chan Su and only 25% protein binding of digoxin, we further demonstrated that positive interference of Chan Su in the FPIA and negative interference of Chan Su in the MEIA of digoxin could be eliminated by monitoring the free digoxin concentration.