This study assessed the N-glycosylation profiles of a full-length trimeric spike protein expressed in either HEK or CHO cells. Glycopeptide analysis was performed using a tandem mass spectrometry workflow and BioPharma Finder™ using HEK and CHO glycan databases for protein characterisation. The results outline important differences in the variety and types of N-glycan generated by the two cell lines across the 22 known N-glycosylation sites of the spike protein. A notable increase in terminal sialylation, as well as the presence of the potentially immunogenic N-glycolylneuraminic acid at a functionally key N-glycosylation site was observed in the CHO derived spike protein. With the potential for the relatively vast and more complex CHO glycan repertoire (182 glycans relative to 39 human glycans) to produce functional implications with CHO expressed spike protein, this study adds valuable knowledge to aid Quality by Design approaches and enable Multi Attribute Monitoring of specific N-glycosylation sites for proteoform analyses. This can further inform antigen development with future variants, to devise updated diagnostic tests and therapeutic vaccine designs