Overall treatment results of chronic hepatitis C have improved markedly with the introduction of pegylated interferon-alpha (PEG-IFN-alpha) and ribavirin combination therapy. However, cure rates in the most common genotype 1 infection are still unsatisfactory. IFN-alpha dose-response studies on viral kinetics suggest that inadequate dosing might be a key factor but drug levels have hardly been tested, which is in part due to difficulties in measuring this cytokine in patient samples. We have shown recently that hepatitis C virus (HCV) replicons are highly sensitive to IFN-alpha. In this report we tested whether the replicon system could be used as a sensitive bioassay to determine the amount of biologically active IFN-alpha in serum or heparinized plasma of patients under therapy. To facilitate the measurements, a stably replicating subgenomic HCV RNA was developed that carries the gene encoding the firefly luciferase. Dose response studies with IFN-alpha demonstrate that the amount of expressed luciferase directly correlates with the level of HCV replication. By using this cell-based assay, serum samples of HCV patients treated with different types and doses of IFN-alpha were analyzed in parallel to IFN-alpha standards made by serial dilutions of the same type of IFN-alpha the patient was treated with. Based on nonlinear logistic models serum concentrations corresponding to 1.3-19 U/ml were determined in patients under standard or high dose IFN-alpha therapy, and from 3.8 to 4.1 ng/ml in patients treated with PEG IFN-alpha. In conclusion, the HCV-replicon based bioassay allows determining the levels of biologically active IFN-alpha in serum and heparinized plasma of patients under treatment.