Amyloid cascades that lead to peptide β-sheet fibrils and plaques are central to many important diseases. Recently, intermediate assemblies of these cascades were identified as the toxic agents that interact with cellular machinery. The location and cause of the transformation from a natively unstructured assembly to the β-sheet oligomers found in all fibrils is important in understanding disease onset and the development of therapeutic agents. Largely, research on this early oligomeric region was unsuccessful because all the traditional techniques measure only the average oligomer properties of the ensemble. We utilized ion-mobility methods to deduce the peptide self-assembly mechanism and examined a series of amyloid-forming peptides clipped from larger peptides or proteins associated with disease. We provide unambiguous evidence for structural transitions in each of these fibril-forming peptide systems and establish the potential of this method for the development of therapeutic agents and drug evaluation.