Protein–protein interactions (PPIs) mediate multiple essential functions and regulatory events in living organisms. The physical interactome of a given protein can be abnormally altered in response to external and internal cues, thus modulating cell physiology and contributing to human pathologies including neurodegenerative diseases. Despite substantial efforts, current approaches cannot pinpoint structure-specific PPIs or their interaction interfaces on a proteome-wide scale. To address this limitation, we have adapted the structural proteomics method known as limited proteolysis–mass spectrometry to identify PPIs by probing protein structural alterations within cellular extracts upon treatment with a protein of interest. We demonstrate the feasibility of our method by analysis of well-characterized PPIs between the respiratory syncytial virus F glycoprotein and its site-specific antibodies. Known antigenic sites were identified directly in complex biological matrices.