AbstractDrug discovery has classically targeted the active sites of enzymes or ligand-binding sites of receptors and ion channels. In an attempt to improve selectivity of drug candidates, modulation of protein–protein interfaces (PPIs) of multiprotein complexes that mediate conformation or colocation of components of cell-regulatory pathways has become a focus of interest. However, PPIs in multiprotein systems continue to pose significant challenges, as they are generally large, flat and poor in distinguishing features, making the design of small molecule antagonists a difficult task. Nevertheless, encouragement has come from the recognition that a few amino acids – so-called hotspots – may contribute the majority of interaction-free energy. The challenges posed by protein–protein interactions have led to a wellspring of creative approaches, including proteomimetics, stapled α-helical peptides and a plethora of antibody inspired molecular designs. Here, we review a more generic approach: fragment-based drug discovery. Fragments allow novel areas of chemical space to be explored more efficiently, but the initial hits have low affinity. This means that they will not normally disrupt PPIs, unless they are tethered, an approach that has been pioneered by Wells and co-workers. An alternative fragment-based approach is to stabilise the uncomplexed components of the multiprotein system in solution and employ conventional fragment-based screening. Here, we describe the current knowledge of the structures and properties of protein–protein interactions and the small molecules that can modulate them. We then describe the use of sensitive biophysical methods – nuclear magnetic resonance, X-ray crystallography, surface plasmon resonance, differential scanning fluorimetry or isothermal calorimetry – to screen and validate fragment binding. Fragment hits can subsequently be evolved into larger molecules with higher affinity and potency. These may provide new leads for drug candidates that target protein–protein interactions and have therapeutic value.