In this report, a novel type I IFN bioassay is presented, which is based on BSL-1-classified VSV replicon particles. The assay is highly sensitive and quantifiable due to the expression of a firefly luciferase reporter gene. In addition, the assay can be rapidly performed within 6 to 7 hours and may be used to determine the antiviral activity of IFNs from humans as well as other species. Thus, this bioassay may be of general interest for all those who want to determine the antiviral activity of cytokines such as type I IFNs.