β-Amyloid peptide (Aβ) is the primary protein component of senile plaques in Alzheimer's disease and is believed to be responsible for the neurodegeneration associated with the disease. Aβ is toxic only when aggregated, however, the size and structure of the aggregated species associated with toxicity is unknown. In the present study, we developed a diffusion-based method to simultaneously separate and detect the biological activity of toxic Aβ oligomers and used the method to examine the relationship between size of aggregated protein and toxicity to SH-SY5Y cells. From these measurements, the effective diffusivity and hydrodynamic radius of the toxic oligomeric species of Aβ could be determined. A sensitivity analysis was performed to examine the effects of model assumptions used in data analysis on the effective diffusivity calculated. The method provides a new estimate of the size of small toxic Aβ species associated with fibril formation. This work contributes to our understanding of the relationship between Aβ structure and toxicity and with further refinements may aid in our ability to design agents which alter the Aβ aggregation/dissociation processes associated with neurotoxicity. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 50–59, 2002.