The interactions of parasites with their different hosts are critical for the completion of each life stage and carbohydrates are important molecules in this process. The roles of protein glycosylation in host-parasite interactions, such as adhesion, invasion, survival and immune system recognition, determine the diversity of outcome of infection. The surface of trypanosomatids is covered by a dense array of heavily glycosylated glycoproteins and glycolipids attached to the membrane via glycosylphosphatidylinositol anchors. These molecules are important in the activation of the innate immune system and initiation of the acquired host immune response in the vertebrate host. Moreover, the remodeling of the parasite surface coat is one key aspect underlying differentiation processes. The study of protein glycosylation in trypanosomatids has unprecedented promise for the discovery of vaccine candidates and for the development of novel chemotherapy approaches and diagnostic tools. However, analytical tools for in depth characterization of site-specific glycan composition and structure linked to proteins still face challenges due to the extensive glycan micro- and macro-heterogeneity. This lecture aims to provide a brief history of the main findings of glycan structures and enzymes involved in Leishmania spp. N- and O-linked protein glycosylation pathways as well as to discuss how analytical methodologies based on mass spectrometry (MS) can be useful as a tool for large-scale characterizing the set of glycans and glycoproteins in parasites. The glycoproteome of three Leishmania species, L. L. amazonensis, L. V. braziliensis and L. L. infantum chagasi, indicated a specie-specific glycosylation which was associated to phylogenetic traits. These methodologies offer novel candidates for biomarker discovery and therapeutics in parasitic diseases.