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). Many small heat shock proteins, including the human sHsp alpha B-crystallin (aBc), 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 aggregation [2], however fundamental questions regarding the molecular mechanism by which polydisperse 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 commonplace and therefore are ideally suited for the study of the interactions between sHsps with misfolded proteins [3]. Therefore, we have recently developed a novel single-molecule fluorescence-based approach to investigate the chaperone action of sHsps. By exploiting this single-molecule fluorescence assay we have, for the first time, determined the stoichiometries of complexes formed between the sHsp aBc and a client protein. By examining the polydispersity and stoichiometries of these complexes over time, we have uncovered unique and important insights into a two-step mechanism by which aBc interacts with misfolded client proteins to prevent their aggregation. Understanding this fundamental mechanism of sHsp action is assisting in addressing current gaps in knowledge that are crucial to understanding how these molecular chaperone function to protect the cell from protein misfolding, as well as their key role in the cellular proteostasis network.