Characterization of biomolecule (e.g., peptides, proteins, oligonucleotides) mixtures is typically quite challenging due to the heterogeneity of the molecules themselves (e.g. due to extensive glycosylation), and/or the matrices in which they are presented to the analytes (e.g. cell culture supernatant). Increasingly, two-dimensional liquid chromatography (2D-LC) methods are being used to improve the depth of characterization of these mixtures, improve the speed of these analyses, or both. This webcast will provide an overview of the key concepts required for successful 2D-LC method development, and several examples of recently developed 2D-LC separations of biomolecules.