We previously developed a biobetter version of rhIFN-β (R27T) that possesses an additional glycosylation site compared with rhIFN-β 1a. Herein, we characterized N-glycosylation heterogeneity of R27T. N-glycan site occupancy manifested as distinct differences in size. The analysis of complex carbohydrate moieties of R27T involved the common biopharmaceutical glycosylation critical quality attributes like core fucosylation, antennary composition, sialylation, lactosamine extensions, linkages, and overall glycan profiles using weak anion exchange and hydrophilic interaction HPLC with 2-aminobenzoic acid-labeled N-glycans. The double-glycosylated form accounted for approx. 94% R27T, while the single-glycosylated form accounted for 6% R27T. N-glycans consisted of a mixture of bi-, tri-, and tetra-antennary glycans, some with lactosamine extensions, but neither outer arm fucose nor α-galactose was detected. Sialic acid major variants, N-acetyl-and N-glycolyl-neuraminic acid were more abundant in R27T than in Rebif. The major N-glycan, accounting for 42% of total N-glycans, had a di-sialylated, core-fucosylated biantennary structure.