Secondary experimental procedures such as immunostaining have been utilized to study wild-type influenza A viruses (IAV) but are inadequate to rapidly determine the virus in infected cells or for the high-throughput screening (HTS) of antivirals or neutralizing antibodies. Reverse genetics approaches have allowed the generation of recombinant IAV expressing bioluminescent (BL) reporters or fluorescent proteins (FPs). These approaches can easily track viral infections in cultured cells and in validated animal models of infection using in vivo imaging systems (IVIS). Here, we describe the experimental procedures to generate recombinant monomeric (m)Cherry-expressing influenza A/Puerto Rico/8/34 (PR8-mCherry) H1N1 by altering the non-structural (NS) vRNA segment and its use in mCherry-based microneutralization assays to assess antivirals and neutralizing antibodies. The experimental procedures could be used for the generation of other recombinant influenza virus types (e.g., influenza B) or IAV subtypes (e.g., H3N2) expressing mCherry or other BL reporters or FPs from the NS or other vRNA segment. These recombinant reporter-expressing viruses represent an excellent toolbox for the identification of prophylactics or therapeutics for the treatment of influenza viral infections in HTS settings as well as to study different aspects related with the biology of influenza viruses and/or its interaction with the host.