Targeting the highly conserved E330 … R416 salt bridge involved in nucleoprotein (NP) trimerization is considered a favorable approach to address the problem of drug resistance in influenza therapy. A photoaffinity probe (1) containing aryl and alkyl azide moieties was synthesized and applied to investigate its binding mode with influenza NP at the trimeric state. Compound 1 possesses appreciable anti-influenza activity with EC50 = 9.1 μM. The photoaffinity labeling experiments were conducted with either the lysate of cells infected by A/WSN/1933(H1N1) virus or the recombinant NP proteins. The aryl azide moiety in bis-azide 1 was selectively activated at 300 nm or longer wavelength to enable covalent linkage with the binding site and adjacent amino acid residues. The remaining alkyl azide group was subjected to Cu(I)-catalyzed alkyne–azide cycloaddition with a biotin-tethered alkyne (20), followed by enrichment using streptavidin magnetic beads. Following trypsin digestion, the labeled peptides were identified by LC-MS/MS analysis and Mascot search. Of the five identified NP peptides, four are associated with the NP-trimer binding pocket. Molecular docking experiments show that the 2-anilino-1,3-thiazole-4-carboxamide backbone of compound 1 occupies almost the same position of E339 … R416 salt bridge to form hydrogen bonds with E339, providing evidence for interference with NP trimerization.