The glycome, that is, the glycan components of a biological source, has been widely reported to change with disease states. However, mining the glycome for biomarkers is complicated by glycan structural heterogeneity. Nanoflow LC, or nano-LC, significantly addresses the problem by providing a highly sensitive and quantitative method of separating and profiling glycans. This review summarizes recent advances in analytical technology and methodology that enhance and augment the advantages offered by nano-LC. (e.g., reversed phase, hydrophilic interaction and porous graphitized carbon chromatography, as well as associated derivatization strategies), detectors (e.g., fluorescence and MS), and technology platforms (particularly chip-based nano-LC) are examined in detail, along with their application to biomarker discovery. Particular emphasis is placed on methods and technologies that allow structure-specific glycan profiling.