Influenza infections are initiated by the binding of the influenza hemagglutinin (HA) and the cellular receptor sialic acids. The binding is followed by internalization, endocytosis, and uncoating to release the influenza genome to the cytoplasm. It is conceivable that specific inhibitors that antagonize any one of these events could prevent the replication of influenza infections. The authors made HA pseudotyped retroviral vectors that express luciferase reporter activities upon transduction to several recipient cells. The transduction of the HA-pseudotype virus particles (HApp) was mediated through the specific interactions between an avian HA and the terminal disaccharides of sialic acid (SA) and galactose (Gal) in alpha-2,3 linkage. The HApp-mediated transduction method was used to develop a high-throughput screening assay and to screen for hits from a fermentation extract library. Specific hits that inhibited the HA-mediated but were noninhibitory to the vesicular stomatitis virus-mediated pseudoviral transductions were identified. A few of these hits have anti-influenza activities that prevent the replication of both H1N1 (WSN) and H5N1 (RG14) influenza viruses.