Changes in protein conformation can affect protein function, but methods to probe these structural changes on a global scale in cells have been lacking. To enable large-scale analyses of protein conformational changes directly in their biological matrices, we present a method that couples limited proteolysis with a targeted proteomics workflow. Using our method, we assessed the structural features of more than 1,000 yeast proteins simultaneously and detected altered conformations for ~300 proteins upon a change of nutrients. We find that some branches of carbon metabolism are transcriptionally regulated whereas others are regulated by enzyme conformational changes. We detect structural changes in aggregation-prone proteins and show the functional relevance of one of these proteins to the metabolic switch. This approach enables probing of both subtle and pronounced structural changes of proteins on a large scale.