•Identification of small molecule inhibitors of protein aggregation using ESI-IMS–MS.•Characterisation of binding mode as negative, specific, non-specific or colloidal.•High throughput ligand screening using ESI-IMS–MS.•Identification of novel small molecule inhibitors of Aβ40 aggregation.Electrospray ionisation-ion mobility spectrometry–mass spectrometry (ESI-IMS–MS) is a powerful method for the study of conformational changes in protein complexes, including oligomeric species populated during protein self-aggregation into amyloid fibrils. Information on the mass, stability, cross-sectional area and ligand binding capability of each transiently populated intermediate, present in the heterogeneous mixture of assembling species, can be determined individually in a single experiment in real-time. Determining the structural characterisation of oligomeric species and alterations in self-assembly pathways observed in the presence of small molecule inhibitors is of great importance, given the urgent demand for effective therapeutics. Recent studies have demonstrated the capability of ESI-IMS–MS to identify small molecule modulators of amyloid assembly and to determine the mechanism by which they interact (positive, negative, non-specific binding, or colloidal) in a high-throughput format. Here, we demonstrate these advances using self-assembly of Aβ40 as an example, and reveal two new inhibitors of Aβ40 fibrillation.Download full-size image