Efforts to develop novel neuraminidase inhibitors (NAIs) for the treatment of influenza are ongoing. Novel NAIs should in particular be also effective against seasonal and/or pandemic N1 that carry a H274Y or N294S substitution (N2 numbering), which are most commonly linked to oseltamivir resistance. Here we report a platform for profiling the efficacy of novel NAIs in the N1 genetic background of influenza A virus.Employing reverse genetics, a set of influenza virus variants containing an amino acid substitution associated with oseltamivir resistance in N1 isolates (H274Y, N294S, Y155H or Q136L) was generated. In parallel, so far unreported mutations of I427 (I427Q and I427M) were investigated. These possibly interfere with the side chain orientation of R371 and alter the binding affinity of most relevant NAIs. The profiling platform was validated with both oseltamivir and zanamivir and exemplarily applied to three analogs with differing decorations at positions 4 and 5. Besides confirming the inhibition profile of zanamivir and oseltamivir, the distinct effect of I427Q/M on the activity of both NAIs was shown. For 5-amidino and 5-guanidino analogs of oseltamivir a significantly stronger inhibition of virus variants carrying a NA-H274Y was confirmed, and additionally shown for NA-N294S and NA-Y155H substitutions as compared to the parent compound. Hence, the herein presented profiling platform is a valid tool for defining the inhibition profile of novel NAIs in the N1 background.