Bovine fetuin is a model protein to study the activity of various glycosidases since it contains both N- and O- glycans attached to the polypeptide chain. We recently showed a novel glycosidase, endo-beta-N-acetylglucosaminidase isolated from an infant gut microbe, Bifidobacterium infantis. This enzyme is capable of cleaving the N-N'-diacetyl chitobiose moiety found in the N-glycan core of a wide variety of proteins. It is considered a promising approach to release N-glycans from complex substrates such as whey proteins due to its high activity and wide substrate specificity. Moreover, it also maintains its activity at high temporal urns enabling the use of this enzyme in thermal dairy processes such as during the pasteurization. Bovine whey is a potential source of glycans providing million tons of glycoproteins annually. Application of EndoBI-1 on bovine whey is challenging due to the complexity of the whey proteins and their O-glycosylation pattern. O-glycans are considered to be a protective agent for N-deglycosylation that hinders the isolation of these recently found novel compounds. In this study, O-glycans were removed from fetuin (both O- and N- glycosylated model glycoprotein) and the contribution of O-glycans to the accessibility of EndoBI-1 to bovine fetuin N-glycans were tested. Released glycans were characterized by advanced mass spectrometry and 22 different N-glycans (including isomers) were monitored. According to the results, it was shown that removing O-glycans from Fetuin increases the Kcat/Km value 0.52 to 1.54 ml/mg x min(-1) and the affinity of EndoBI-1 (Km value from 0.32 to 0.22 mg/ml) to target N-glycans enabling more feasible application of this enzyme in dairy streams.