A glycoprotein is a glycoconjugate in which a protein has one or more glycans covalently attached to a polypeptide backbone, usually via N- or O- linkages. This modification can affect the structure, function, interaction, and folding of proteins. Glycosylation is crucial for physiological and pathological cellular functions.1 Specific N- and O-linked glycoprotein changes are associated with disease progression and the identification of these glycoproteins has potential for use in disease diagnostics, prognosis, and treatment.2,3 For example, Immunoglobulin G (IgG), the most common serum antibody, is N-glycosylated and studies have established IgG glycosylation as a key regulator of humoral immune activity.4,5,6,7 The biological significance of IgG and other glycoproteins makes the development of accurate methods to analyze them vital. Structural analysis of glycoproteins is generally performed with or without release of the glycan modification. A proteolytic digestion can be performed on a glycoprotein to produce glycopeptides. In addition, N-glycans can be released by Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase (PNGase F). Liquid chromatography paired with mass spectrometry (LC-MS) is commonly used for the analysis of proteins, glycoproteins, and glycans. The emergence of Tandem MS (MS/MS) and hydrophilic interaction liquid chromatography (HILIC) methodologies have assisted in the identification and characterization of glycoproteins. The current work presented here utilizes HILIC-LC-MS methods to address various gaps in the field by increasing the depth of information obtained from experiments, including investigating retention behavior, reducing the identification of false positives, and determining kinetics of PNGase F deglycosylation for complete glycan release. The purpose of the work presented here is multifaceted: first, to use HILIC to analyze the separation of O-mannose glycosylated peptides and investigate their retention behavior to facilitate faster, and more confident identification and characterization of unknowns. Second, a novel HILIC-LC-MS method, n-Asp and i-Asp to reduce false positives (NIFP), was proposed to reduce interferences in glycosylation site mapping.8 Third, the kinetics of PNGase F deglycosylation reactions performed on intact and trypsin digested IgGs were investigated for the development of efficient methods for accurate quantitation. The effect of different PNGase F enzyme preparations on the kinetics of deglycosylating Human Serum IgGs was also investigated.