Achieving the successful separation and analysis of amyloid and other large protein aggregates can be a difficult proposition. Field-flow fractionation (FFF) is a flow-based separation method like chromatography; however, FFF is capable of high-resolution separations in the absence of a stationary matrix. Thus, FFF is a relatively gentle technique and is well suited to the task of separating large macromolecules and macromolecular complexes. Flow field-flow fractionation (FlFFF), one of the techniques in the FFF family, has been used to successfully fractionate a wide size range of prion protein aggregates, allowing their subsequent characterization by several biophysical and biochemical methods. The ability to easily adjust the strength of the field used during separation means that FlFFF could be applied to particles ranging from 1 nm to nearly 100 mum in size. This flexibility, coupled with the ability to produce fast, high-resolution separations, makes FFF a potentially valuable tool in the field of amyloid research.