Protein–protein interactions are essential in every aspect of cellular activity. Multiprotein complexes form and dissociate constantly in a specifically tuned manner, often by conserved mechanisms. Protein domains that bind proline‐rich motifs (PRMs) are frequently involved in signaling events. The unique properties of proline provide a mechanism for highly discriminatory recognition without requiring high affinities. We present herein a detailed, quantitative assessment of the structural features that define the interfaces between PRM‐binding domains and their target PRMs, and investigate the specificity of PRM recognition. Together with the analysis of peptide‐library screens, this approach has allowed the identification of several highly conserved key interactions found in all complexes of PRM‐binding domains. The inhibition of protein–protein interactions by using small‐molecule agents is very challenging. Therefore, it is important to first pinpoint the critical interactions that must be considered in the design of inhibitors of PRM‐binding domains.