Small heat shock proteins (sHsps) are a family of ubiquitous intracellular molecular chaperones that are up-regulated under stress conditions and play a vital role in protein homeostasis (proteostasis). The sHsps exist as large polydisperse assemblies of up to 1 MDa in size that undergo rapid subunit exchange 1. It is commonly accepted that these chaperones work by trapping misfolded proteins to prevent their aggregation2, however, fundamental questions regarding the molecular mechanism by which sHsps interact with these misfolded proteins remain unanswered. This is because these chaperones are notoriously difficult to study using bulk analysis techniques due to the dynamic and transient nature of species formed during their interactions with aggregation-prone proteins. However, over the past two decades the use of single-molecule techniques to study dynamic protein complexes has become more commonplace3. By observing proteins one-by-one, rare, transient and dynamic species can be characterised in great detail. These capabilities make single-molecule methods ideally suited to the study of the interactions of sHsps with misfolded proteins. Therefore, we have developed a single-molecule fluorescence microscopy-based approach for the study of the sHsps and aggregation-prone proteins. Using this approach we have observed the interaction of the sHsps Hsp27 and aB-crystallin with aggregation-prone proteins. Furthermore we have characterised and determined the stoichiometries of complexes formed in solution between sHsps and aggregation-prone proteins at a single-molecule level. Overall, addressing these crucial gaps in knowledge will contribute to our understanding of the molecular mechanisms by which sHsps function as molecular chaperones, thus preserving cellular proteostasis.