Proteomics has been broadly applied to detect changes in protein levels in response to perturbations and derive information on altered pathways. Beyond protein expression changes, however, biological processes are also regulated by molecular events such as intermolecular interactions, chemical modification and conformational changes. These events do not affect protein levels and therefore escape detection in classical proteomic screens. I will present a new global readout of protein structure that detects various types of protein functional alterations concomitantly. The approach, relying on the LiP-MS technique, monitors protein structural changes in thousands of proteins simultaneously and in situ. It captures enzyme activity changes, allosteric regulation, phosphorylation and protein complex formation and pinpoints regulated functional sites, thus supporting the generation of mechanistic hypotheses. I will present the application of this strategy to the study of complex cellular phenotypes. Further, I will show how LiP-MS can be used to identify targets of small molecules and binding sites in an unbiased manner and directly from cell and tissue extracts.