The liver-derived corticosteroid-binding globulin (CBG) is a heavily N-glycosylated transport protein of anti-inflammatory cortisol in human plasma. We have previously demonstrated that the Asn347-glycosylation of the exposed reactive centre loop (RCL) of human CBG impacts the delivery mechanism of cortisol by modulating the neutrophil elastase cleavage rate. However, the structure-function relationship of this complex N-glycoprotein remains largely unexplored. Recombinant human CBG variants may be valuable in aiding our understanding of CBG glycobiology. To this end, the glycosylation of newly available HEK293-expressed recombinant human CBG products including polyhistidine- (His-CBG) and biotin-tagged (biotin-CBG) forms were here site-specifically profiled using mass spectrometry-based glycomics and glycopeptide analysis. Glycomics profiling indicated that both the His- and biotin-CBG carry similar, but not identical, N-glycosylation despite their common expression system. Complex-type core-fucosylated bi-, tri-, and even higher antennary sialoglycans were identified, glyco-features that are recapitulated in native human CBG. Detailed glycopeptide analyses involving multiple protease digestion strategies, glycopeptide enrichment, and orthogonal dissociation methods used with advanced mass spectrometry detection were employed to site-specifically determine several glyco-features unique to His- and biotin-CBG including the presence of GalNAc(β1,4)GlcNAc (LacdiNAc) and NeuAc(α2,3/6)GalNAc(β1,4)GlcNAc N-glycan motifs, and oligomannosidic N-glycans with and without mannose-6-phosphate. Interestingly, the glycoprofiling also demonstrated the presence of O-glycosylation of both His- and biotin-CBG, a feature not previously reported for native human CBG. Importantly, the two identified O-glycans (Gal1GalNAc1NeuAc2 and Gal2GalNAc2NeuAc2) could be located to the RCL region in proximity to the neutrophil elastase cleavage site and hence may impact cortisol delivery. In conclusion, this site-specific glycoprofiling study has accurately documented the glycosylation of two recombinant human CBG variants, an important prerequisite for future glycobiological and structure-function studies using recombinant human CBG.