Glycosylation can have a profound influence on the function of a variety of eukaryotic cells. In particular, it can affect signal transduction and cell-cell communication properties and thus shape critical cell decisions, including the regulation of differentiation and apoptosis. Regulation of glycosylation has multiple layers of complexity, both structural and functional, which make its experimental and theoretical analysis difficult to perform and interpret. Novel research methodologies provided by systems biology can help to address many outstanding issues and integrate glycosylation with other metabolic and cell regulation processes. Here we review the toolbox available for biochemical systems analysis of glycosylation.