Development of a thorough understanding of the solution polydispersity of therapeutic glycoproteins including monoclonal antibodies is an important and challenging undertaking. Degradation pathways involving fragmentation could result in loss of therapeutic efficacy. Protein aggregation on the other hand is frequently considered a critical quality attribute, and concerns exist that protein aggregates could result in undesirable immunological consequences (1). Sedimentation velocity analysis performed in the analytical ultracentrifuge (SV-AUC) provides a uniquely powerful first principal measure of the hydrodynamic size and shape of proteins under conditions that can come very close to the formulated drug product. This technique avoids the potential pitfalls associated with size exclusion chromatography (SEC) including on-column dilution, adsorption or disruption of species by a stationary phase, and the need to use high ionic strength mobile phases to screen unwanted electrostatic interactions (2, 3). Furthermore, not only does SV-AUC provide a quantitative size distribution analysis, but it also provides information about macromolecular conformation. For these reasons, use of SV-AUC for analysis of therapeutic monoclonal antibodies has become widespread throughout the biopharmaceutical industry and is one of the most common orthogonal techniques to SEC for measuring aggregate and fragment levels (4–9). The studies outlined in this chapter describe the basic principles of designing, collecting, and analyzing experimental data using SV-AUC with a focus on methods for therapeutic monoclonal antibodies and other similar biologics. Details are given that facilitate the acquisition of high quality data sets that in turn simplify data analysis resulting in robust and accurate measures of solution polydispersity.