Influenza A virus (IAV) infection poses a serious health threat and novel antiviral strategies are needed. Defective interfering particles (DIPs) can be generated in IAV infected cells due to errors of the viral polymerase and may suppress spread of wild type (wt) virus. The antiviral activity of DIPs is exerted by a DI genomic RNA segment that usually contains a large deletion and suppresses amplification of wt segments, potentially by competing for cellular and viral resources. DI-244 is a naturally occurring prototypic segment 1-derived DI RNA in which most of the PB2 open reading frame has been deleted and which is currently developed for antiviral therapy. At present, coinfection with wt virus is required for production of DI-244 particles which raises concerns regarding biosafety and may complicate interpretation of research results. Here, we show that cocultures of 293T and MDCK cell lines stably expressing codon optimized PB2 allow production of DI-244 particles solely from plasmids and in the absence of helper virus. Moreover, we demonstrate that infectivity of these particles can be quantified using MDCK-PB2 cells. Finally, we report that the DI-244 particles produced in this novel system exert potent antiviral activity against H1N1 and H3N2 IAV but not against the unrelated vesicular stomatitis virus. This is the first report of DIP production in the absence of infectious IAV and may spur efforts to develop DIPs for antiviral therapy.