The development of pegylated interferons (PEG-IFN) has significantly improved the eradication rates in patients with chronic hepatitis C. Two forms of PEG-IFN have been developed, based on two pegylation chemistries: the 12-kDa linear PEG-IFN-alpha2b and the 40-kDa branched PEG-IFN-alpha2a. We compared the in vitro antiviral activity of linear and branched PEG-IFN using the vesicular stomatitis virus (VSV) cytopathic effect (CPE) reduction assay. The specific antiviral activity of branched PEG-IFN was 7% of that of linear PEG-IFN. A given quantity of linear and of branched PEG-IFN does not represent the same biologic activity. A bioassay could give new insights to compare the pharmacokinetic profile of linear PEG-IFN and of branched PEG-IFN.