AbstractMany analytical challenges in biomedicine arise from the generally high heterogeneity and complexity of glycan‐ and glycoconjugate‐containing samples, which are often only available in minute amounts. Therefore, highly sensitive workflows and detection methods are required. In this review mass spectrometric workflows and detection methods are evaluated for glycans and glycoproteins. Furthermore, glycomic methodologies and innovations that are tailored for enzymatic treatments, chemical derivatization, purification, separation, and detection at high sensitivity are highlighted. The discussion is focused on the analysis of mammalian N‐linked and GalNAc‐type O‐linked glycans.