Abstract Disopyramide is an antiarrythmic drug with concentration dependent protein binding within the therapeutic range. We found good agreement between fluorescence polarization immunoassay (FPIA, Abbott TDX) and high performance liquid chromatography (HPLC) for total disopyramide among 27 admission patients' sera (FPIA = 1.12 (HPLC) −0.002, r = 0.982, SEE = 0.378 mg/l). Agreement between FPIA and HPLC for free disopyramide was similarly good (FPIA = 0.867 (HPLC) −0.003, r = 0.986, SEE = 0.09 mg/l, n = 27). Serum α 1 acid glycoprotein concentration (AAG) and free fraction (FF) percent of disopyramide correlated inversely (FF = −0.0112 (AAG) + 31.3 r = 0.707, SEE = 6.41 mg/l, n = 24), and the free disopyramide fraction varied greatly. For two pooled sera with 12 different disopyramide concentrations (range from 0.5–20.0 mg/l), the proportion of free fraction ranged from 6.5 to 73.2%. Overall, we found the free disopyramide fraction variable with each individual, total drug, and protein concentration. Therefore, free drug concentration should be monitored in disopyramide therapy, and FPIA is reliable for free as well as total drug assay.