Despite the immense importance of enzyme-substrate reactions, there is a lack of generic and unbiased tools for identifying the molecular components participating in these reactions on a cellular level. Here we developed a universal method called System-wide Identification of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that enzymatic post-translational modification of substrate proteins changes their thermal stability. SIESTA successfully identified several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, as well as poly-(ADP-ribose) polymerase-10 and protein kinase B (AKT1) systems. A number of putative substrates for each enzyme system were confirmed by targeted mass spectrometry and functional assays. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, open new opportunities in investigating the effect of PTMs on protein stability, and facilitate drug discovery.