Sedimentation velocity analytical ultracentrifugation is a powerful classical method to study protein self-association processes in solution based on the size-dependent macromolecular migration in the centrifugal field. This technique can elucidate the assembly scheme, measure affinities ranging from picomolar to millimolar Kd , and in favorable cases provide information on oligomer lifetimes and hydrodynamic shape. The present step-by-step protocols detail the essential steps of instrument calibration, experimental setup, and data analysis. Using a widely available commercial protein as a model system, the protocols invite replication and comparison with our results. A commentary discusses principles for modifications in the protocols that may be necessary to optimize application of sedimentation velocity analysis to other self-associating proteins. ©2020 Wiley Periodicals LLC. Basic Protocol 1: Measurement of external calibration factors Basic Protocol 2: Sedimentation velocity experiment for protein self-association Basic Protocol 3: Sedimentation coefficient distribution analysis in SEDFIT and isotherm analysis in SEDPHAT.