In this study, an HPLC HILIC-UV method was developed for the analysis of intact neo-glycoproteins. During method development the experimental conditions evaluated involved different HILIC columns (TSKgel Amide-80 and ZIC-pHILIC), and water-acetonitrile mixtures containing various types of acids and salts. The final selected method was based on a TSKgel Amide-80 column and a mobile phase composed of acetonitrile and water both containing 10 mM HClO4. The influence of temperature and sample preparation on the chromatographic performances of the HILIC method was also investigated. The method was applied to the separation of neo-glycoproteins prepared starting from the model protein RNase A by chemical conjugation of different glycans. Using the method here reported it was possible to monitor by UV detection the glycosylation reaction and assess the distribution of neo-glycoprotein isoforms without laborious sample workup prior to analysis.