Glycosylation is the most frequent post-translational modification of proteins. However, characterization of glycoproteins remains elusive due to their poor receptivity to current structural methods and difficulties in sample preparation. Here, we use (¹H,¹⁵N) nuclear magnetic resonance (NMR) to characterize structural perturbations of the enzyme, Ribonuclease B (RNase B), relative to its pre-glycosylated form, RNase A. RNase is an excellent model system for the study of glycoproteins because of significant previous work on RNase A catalysis, structure and dynamics. Much less is known about RNase B. The oligosaccharide attaches at the non-catalytic Asn34, over 15 Å from the active site. Its presence is known to reduce catalytic activity in RNase B, but the mechanism of this modulation is not understood. Previous (¹H, ²H) exchange NMR revealed overall rigidification of RNase B relative to A [Joao & Dwek, Eur. J. Biochem. (1993)], while other studies demonstrated the importance of structural flexibility to RNase A catalysis [Cole & Loria, Biochemistry (2002)]. To explore lost activity in terms of the reduced motional freedom in RNase B, we characterized differential A and B structure using natural-abundance ¹⁵N samples for two-dimensional (¹H,¹⁵N) NMR of RNase B. We have assigned ~80% of the amide resonances by comparison with temperature-dependent RNase A chemical shifts. Differential RNase A-B shifts indicate minimal structural perturbations to the glycoprotein backbone. The absence of changes supports our hypothesis that reduced RNase B flexibility contributes to functional losses. We present strategies for future elucidation of RNase B dynamics via spin-relaxation NMR.