Thiopurines are the cornerstone of treatment for a wide variety of medical disorders, ranging from pediatric leukemia to inflammatory bowel disease. Because of their complex metabolism and potential toxicities, the use of biomarkers to predict risk and response is paramount. Thiopurine S-methyltransferase and thiopurine metabolite levels have emerged as companion diagnostics with crucial roles in facilitating safe and effective treatment. This review serves to update the reader on how these tools are being developed and implemented in clinical practice. A useful paradigm in thiopurine therapeutic strategy is presented, along with fresh insights into the mechanisms underlying these approaches. We elaborate on potential future developments in the optimization of thiopurine therapy.