Updated publication reference for PubMed record(s): 33637753. 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, and applies the concept of specificity to reveal potential substrates. We have already shown the applicability of SIESTA in substrate discovery for TXNRD1 and PARP10 enzymes. SIESTA successfully identified several known and novel substrate candidates for protein kinase B (AKT1). A number of putative substrates were confirmed by 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.