<p>Protein–protein interactions (PPIs) play a compelling role in biological systems. Additionally, they are promising therapeutic targets for various diseases. However, the design of small molecule inhibitors for PPIs is challenging due to the dynamic and featureless nature of the PPI interfaces, which often lack well-defined binding pockets. To address this challenge, the PPIscout approach employs mixed solvent molecular dynamics (MD) simulations to identify binding hotspots at PPI interfaces. This technique utilizes mixed amino acid–water MD simulations with capped amino acids as probe molecules. This method has been applied to three target proteins: MDM2, Bcl-XL and PDZ6. The mixed solvent simulations revealed that the probe molecules predominantly accumulated at the PPI interfaces, highlighting potential hotspots. These findings are corroborated with results from FTMap, a computational solvent mapping tool. Results demonstrate that mixed amino acid–water MD simulations effectively identify PPI hotspots and favorable interactions that are crucial for drug design. Moreover, the method also unveils potential allosteric sites that could be targeted for the development of peptides or small molecule drugs.</p> Graphical abstract <p></p> <p> PPIscout employs mixed-solvent molecular dynamics (MD) simulations, using amino acid probes, to identify binding hotspots and potential allosteric sites on dynamic protein-protein interaction (PPI) interfaces. This computational method reveals crucial interaction sites, aiding in the design of peptide and small molecule inhibitors.</p>