Conjugation chemistry is one of the main parameters affecting immunogenicity of glycoconjugate vaccines and a rational approach toward a deeper understanding of their mechanism of action will greatly benefit from highly-defined and well-characterized structures. Herein, different conjugation methods were investigated with the aim of controlling glycosylation site and glycosylation density on the carrier protein. S. Typhimurium lipopolysaccharide O-Antigen and CRM₁₉₇ carrier protein were used as models. In particular, thiol and click chemistry were examined, both involving the linkage of the terminal reducing sugar unit of the O-Antigen chain to different amino acids on the carrier protein. Thiol chemistry allowed O-Antigen conjugation only when the carrier protein was activated on the lysines and with a relative high number of linkers, while click chemistry allowed conjugate generation even when just one position on the protein was activated and to both lysine and tyrosine sites. The study highlights click chemistry as a leading approach for the synthesis of well-defined glycoconjugates, useful to investigate the relationship between conjugate design and immune response.