Glycosylation is a common post-translational modification that confers important properties to proteins. Glycans are implicated in a wide range of intracellular, cell-cell and cell-matrix recognition events and, therefore, are of great biological interest. In this paper we have taken an enzymatic approach by using peptide N-glycosidase F to release N-linked glycans from ovomucoid. Our study revealed that the protein in its native conformation was susceptible to this enzyme. Nevertheless, denaturation by means of both heat (at 100 °C) and reducing agent treatment, before the enzymatic exposure, greatly enhanced the extent of deglycosylation.SDS-PAGE and Western blot analyses revealed that the intact protein migrates as a diffuse band that was attributed to glycosylation heterogeneity. The complete deglycosylation of the protein resulted in a shift of the protein migration and in the formation of a sharp protein band and it was confirmed by gel filtration, FTIR and MALDI-TOF analyses. Importantly, LC-MS/MS revealed for the first time the presence of eight potential deglycosylation sites on the protein.