Alpha-1-acid glycoprotein (AGP) is an acute-phase plasma protein with high carbohydrate content attached in the form of five N-linked complex glycans. Altered glycosilation of AGP occurs in many physiological and pathophysiological conditions. Recently, a cost-effective method for a high-throughput detailed AGP N-glycosylation profiling was developed, which provides site-specific glycosylation information from a small volume of blood serum or plasma. Using the method, it was demonstrated that N-glycan profile of AGP is stable in a healthy individual and that changes in N-glycan profile could help distinguish individuals who are at risk of type 2 diabetes. AGP is enriched from blood plasma by acid precipitation of seromucoid fraction, after which it undergoes reduction, alkylation and trypsinization to be further enriched using solid-phase extraction (HILIC-SPE). Glycopeptides obtained by this process are then analysed by reversed-phase liquid chromatography paired with electrospray ionization-MS. During the implementation of the method on large cohorts, reduced intensity of glycosylation site III signal was observed in a certain number of measurements. To determine the cause of irregularity, influence of trypsin concentration and increased concentration of acetonitrile in HILIC mobile phase on intensity of glycosylation site III signal was evaluated. The effect of trypsin was evaluated due to AGP being resistant to conventional tryptic digestion, whereas a higher concentration of ACN was investigated because of an extensive amount of hydrophobic peptide moiety of glycosylation site III which could lower the affinity towards the stationary phase in HILIC-SPE enrichment The change in trypsin concentration didn't affect the intensity of the signal, whereas a higher concentration of ACN has enhanced the signal intensity of the glycosylation site of interest. These changes in the protocol (except for the trypsin concentration that was five times higher than the standard) have resulted in decreased precision compared to the standard method. This decrease is not suitable for the method optimisation. In further research, other stationary phases that could be suitable for HILIC-SPE enrichment or different enrichment methods should be tested in order to find a method that would also meet the precision criteria while increasing the signal intensity of the glycosylation site III.