A modified EMIT 2000 digoxin assay was developed on the Cobas Mira plus analyzer for the determination of very low plasma concentrations of the drug. The major modifications were a higher plasma volume withdrawn during the analysis step and calibration curves constructed in the range 0-2 ng/ml using calibrators made up with biological matrix. Assays were controlled with an internal, four-level quality control (targets: 0.15; 0.60; 1.70; 2.70 ng/mL). The within-day and day-to-day mean observed values ± SD (n = 10) of these quality controls were 0.14 ± 0.02 and 0.15 ± 0.02 ng/mL; 0.57 ± 0.01 and 00.64 ± 0.03 ng/mL; 1.55 ± 0.06 and 1.62 ± 0.04 ng/mL, 2.82 ± 0.09 and 2.82 ± 0.12 ng/mL, respectively. The detection and the quantification limits were 0.02 and 0.08 ng/mL, respectively. No significant difference was observed between digoxin plasma concentrations measured by the original and the modified EMIT 2000 digoxin assay in 25 plasma specimens, ranging from 0.4 to 3.0 ng/mL, from patients receiving the drug. This modified digoxin EMIT 2000 assay was subsequently used to study digoxin pharmacokinetics after each of 18 healthy volunteers was administered a single 0.5 mg oral dose. The pharmacokinetic parameters found in this study were in accordance with the literature in healthy subjects, using radioimmunoassay (RIA) for digoxin plasma concentration determinations. In conclusion, the lower limit of quantification of this modified EMIT 2000 digoxin assay is similar to that of RIA and can serve as a valuable screen for digoxin pharmacokinetic interactions studies.