B7 immune checkpoint proteins play an important role in modulating antitumor immune response. These proteins interact with co-inhibitory or co-stimulatory receptors on immune and tumor cells, and their expression associates with cancer progression and poor prognosis. B7 proteins are highly glycosylated, as part of their posttranslational modification process. This process consists of the covalent addition of glycans to proteins, which directly affects protein expression, stability, localization, and interaction with partners. Protein glycosylation of immune checkpoint proteins has also been linked to therapy response and immune evasion. Here, we describe experimental methodologies to study the molecular and clinical impact of glycosylation on the B7 family of glycoproteins. We summarize experimental protocols and technical notes to assess the N-glycosylation of B7 proteins, and to study the functional effect of glycosylation on the expression and localization of B7 proteins.