Immunogenicity is the ability of a therapeutic to provoke an undesired immune response and is considered a primary concern during the development of biosimilars and new drug products. Recombinant human Interferon alpha 2b (IFN) is widely used for the treatment of viral diseases such as chronic Hepatitis B and C (CHC). In an attempt to prolong its plasma half-life, hyperglycosylated derivatives with different glycosylation patterns and remarkable pharmacokinetic profiles were developed and designated as IFN-2NM47/95, IFN-3NM47, IFN-3NM47/95 and IFN- 3NM47-Nter. However, there is growing evidence showing that repeated dosing of IFN over several months induces neutralizing antibodies (NAb) against the therapeutic in up to 80% of patients, depending on the indication. Moreover, type I IFNs can both induce and unmask sub-clinical autoimmune diseases. For this, the aim of this study was to investigate the immunogenicity of these new hyperglycosylated IFN variants through a comparative ex vivo study that also included the unmodified version or wild type (IFN-WT). Blood samples were taken from 12 healthy donors and peripheral blood mononuclear cells (PBMC) were isolated from each donor by Ficoll-Hypaque density centrifugation. HLA-DR1 allotypes were determined by Luminex technology. Monocyte derived-dendritic cells (DCs) were generated and used to endocyte and process the IFN variants. Then, antigen-pulsed DCs were incubated with autologous T-cells. After 2-3 days of incubation, supernatants were collected and analyzed for IFN-gamma and IL-4 productions by sandwich ELISA. A stimulation index (SI) criteria was defined as the ratio of cytokine concentrations from protein challenged treated PBMCs and unchallenged PMBCs (culture media) and a geometric mean (GM) was then calculated. Positive responses were defined by donors who produced a SI higher than GM. The T-cell proliferation assays showed that all tested proteins induced exclusively IFN-gamma secretion, but in different levels depending on the protein and the donor. In particular, HLA-DRB1*08, HLA-DRB1*09, HLA-DRB1*13 and HLA-DRB1*16 alleles were directly involved in the IFN-derived peptide presentation. Also, a comparative analysis revealed that 3NM47/95 was the most immunogenic IFN version with 42% of positive responses. In contrast, IFN-3NM47-Nter was the variant which exhibited the lower immunogenicity (25% of responders). In addition, a similar proportion of responders (33%) was observed for IFN-2NM47/95, IFN- 3NM47 and IFN-WT. It is important to highlight that IFN-3NM47-Nter contain an additional glycosylation site when compared with 3NM47/95. Therefore, this suggests that higher glycan contents played a role in antigen recognition, processing and/or presentation. Considering the reduced immunogenicity for IFN- 3NM47-Nter observed here and its superior pharmacokinetic properties, altogether these results highlight IFN-3NM47-Nter as a promising candidate for clinical use in antiviral therapy.