Influenza A virus has been a constant threat to human population causing four major pandemics. In 2009 during a pandemic, a novel H1N1 viral strain emerged (H1N1pdm09), demonstrating resistance to the antiviral drug oseltamivir due to a single amino acid substitution H275Y in neuraminidase (NA). This resistance came at a small cost of the viral fitness, yet secondary permissive mutations such as V241I/N369K were reported to restore NA’s activity to the levels of that of in the wild-type virus (1). NA is a homotetramer glycoprotein consisting of a catalytic head domain with four active sites and a stalk region that anchors NA to the virion viral membrane. Both domains contain a number of glycosylation sites that have been reported to significantly affect the pathogenicity (2), the efficient incorporation and replication of the virus in host cells, (3) as well as the structural stability of NA itself (4). However, how the glycosylation sites were affected in the new H1N1pdm09 strain remains elusive. Considering the central role of NA in maintaining influenza’s transmissibility together with the evidence suggesting glycosylation alternations could implicate with NA’s biological activity (2), we aim to characterise the glycosylation profile of the NA H275Y and to assess whether permissive mutations induce any changes. Here, we reproduced the mutations in the NA (A/H1N1/Auckland/2009) via site-directed mutagenesis PCR and expressed the recombinant soluble NAs (rNA) in HEK293T cells. Using the Waters Select Series cyclic ion mobility mass spectrometer and the SCIEX ZenoTOF mass spectromerter we successfully characterised the glycosylation nuances between the variable rNAs under CID and EAD fragmentation modes. Taking into consideration the accumulative evidence that constitute NA an important target for the development of next-generation flu vaccines (5), the results of this study highlight exciting future therapeutic avenues.