Influenza A viruses often cause seasonal respiratory diseases, and even lead to severe global pandemics. Nucleoprotein (NP) is regarded as a druggable target due to its conserved sequence and important functions during influenza virus life cycle. Wong and coworkers have found a lead compound A, which has good inhibitory activity against influenza viruses by disrupting the E339...R416 salt bridge interaction between NP monomers. Based on the structure of this lead compound, our group currently involves in the structure−activity relationship (SAR) studies. My work is to verify the target protein of H1N1 virus by using a combination of techniques including photoaffinity labeling and mass spectrometry. Based on the SAR study, a bifunctional compound is designed to bear both aryl azide and alkyl azide moieties. The aryl azide moiety can be irradiated by UV light to produce an active nitrene intermediate, which will form covalent bonds with proximal residues in the active site of target protein. The alkyl azide moiety is located on the opposite end to react with a biotin tag through the Cu(I)-catalyzed alkyne−azide cycloaddition (CuAAC) for protein enrichment and analysis. This bifunctional compound was synthesized, and shown to possess good inhibitory activity (EC50 = 9.06 uM) against influenza virus. The photoaffinity labeling experiment and protein identification were performed and the result allows us to further explore the binding mode of drug candidate with influenza virus.