Efficient approaches that can help to select vaccine strains for the influenza virus neuraminidase (NA) antigen are currently needed to advance the development of vaccines containing NA. Here, we present a rapid and cost-effective solution-based NA active site proximity assay (NASPA) for measuring NA activity inhibitory (NAI) antibodies. This simplified assay uses large “bulky” NA active site-binding inhibitors to replace the sialylated glycoprotein substrates in common NA enzyme-linked lectin assay (ELLA) approaches. Our results with ferret antisera and monoclonal antibodies against vaccine strain NAs show a strong correlation between NASPA and ELLA titers, and that NASPA titers are not influenced by anti-HA antibodies. Consequently, NASPA can be used with influenza A or B strains and with the latter it revealed incremental antigenic changes in the NAs from recent B Victoria lineage vaccine strains. By coupling NASPA with a simple activity assay, we also found that steric and active site-binding NAI antibodies against circulating NAs are common in adult human sera. Finally, we demonstrate that NASPA can be modified by incorporating novel NA substrate-analog-based inhibitors. Together, these results suggest that NASPA can aid the development of vaccines containing NA by helping to select suitable vaccine strains and profile anti-NA antibody responses.