Viruses can evade the host immune system by displaying numerous glycans on their surface ‘spike-proteins’ that cover immune epitopes. We have developed an ultrasensitive ‘single pot’ method to assess glycan occupancy and the extent of glycan processing from high-mannose to complex forms at each N-glycosylation site. Though aimed at characterizing glycosylation of viral spike-proteins as potential vaccines, this method is applicable for analysis of site-specific glycosylation of any glycoprotein.