Although size exclusion chromatography (SEC) has been, and will continue to be, the primary analytical tool for characterization of the content and size distribution of non-particulate aggregates in protein pharmaceuticals, regulatory concerns are driving increased use of alternative and complementary methods such as analytical ultracentrifugation and light scattering techniques. This review will highlight and critically review the capabilities, advantages, and drawbacks of SEC, analytical ultracentrifugation, and light scattering methods for characterizing aggregates with sizes below about 0.3 microns. The physical principles of the biophysical methods are briefly described and examples of data for real samples and how that data is interpreted are given to help clarify capabilities and weaknesses.