Aggregation is a major degradation pathway that needs to be characterized and controlled during the development of protein pharmaceuticals. Analytical ultracentrifugation-sedimentation velocity (AUC-SV) is emerging as an important orthogonal tool to size exclusion chromatography to quantitate aggregates. However, the precision and accuracy of modern AUC-SV and the experimental variables that influence these two performance parameters need to be better understood and controlled. To understand the impact of experimental and data analysis variables on the precision, aggregate content in monoclonal antibody preparations was measured by AUC-SV and analyzed by the software program Sedfit. Accuracy and limit of detection were evaluated by spiking a known quantity of a sample enriched in aggregate fraction. The results suggest experimental and data analysis approaches that improve precision and accuracy of aggregate quantitation by AUC-SV. Both precision and accuracy were found to be highly dependent on the quality of the centerpieces as assessed by microscopic examination. The level of precision for quantitating aggregates was found to be approximately +/-0.3 to 0.7% over the aggregate content range of approximately 0.6 to 67%. Accuracy, as indicated by approximately 80 to 90% spiked recovery, could be achieved down to aggregate levels as low as approximately 1.5%, whereas the limits of detection and quantitation appear to be approximately 0.2 and 0.6%, respectively.