Protein structural changes induced by external perturbations or internal cues can profoundly influence protein activity and thus modulate cellular physiology. A number of biophysical approaches are available to probe protein structural changes, but these are not applicable to a whole proteome in a biological extract. Limited proteolysis-coupled mass spectrometry (LiP-MS) is a recently developed proteomics approach that enables the identification of protein structural changes directly in their complex biological context on a proteome-wide scale. After perturbations of interest, proteome extracts are subjected to a double-protease digestion step with a nonspecific protease applied under native conditions, followed by complete digestion with the sequence-specific protease trypsin under denaturing conditions. This sequential treatment generates structure-specific peptides amenable to bottom-up MS analysis. Next, a proteomics workflow involving shotgun or targeted MS and label-free quantification is applied to measure structure-dependent proteolytic patterns directly in the proteome extract. Possible applications of LiP-MS include discovery of perturbation-induced protein structural alterations, identification of drug targets, detection of disease-associated protein structural states, and analysis of protein aggregates directly in biological samples. The approach also enables identification of the specific protein regions involved in the structural transition or affected by the binding event. Sample preparation takes approximately 2 d, followed by one to several days of MS and data analysis time, depending on the number of samples analyzed. Scientists with basic biochemistry training can implement the sample preparation steps. MS measurement and data analysis require a background in proteomics.