Abstract: Target identification of bioactive molecules is important for deciphering their functions and action mechanism in cells, as well as for drug development. Thermal proteome profiling (TPP), based on the principle that protein-ligand binding can increase the thermal stability of proteins, is a new technology for identifying target proteins of bioactive molecule. One of the advantages of TPP is that it doesn’t require functionalization of a bioactive compound, which significantly broadens its application in drug discovery and development. TH588 is an inhibitor of 7,8-dihydro-8-oxoguanine triphosphatase (MTH1), and it inhibits the proliferation of tumor cells by increasing the oxidative damage of DNA. However, previous studies showed that TH588 also executed MTH1-independent anti-tumor effects on multiple cancers by depolymerizing tubulin, increasing oxidative damage, and inactivating PI3K-AKT-mTOR signaling pathway. Therefore, identification of off-targets for TH588 is essential for understanding TH588 functions and its further chemical modification in drug development. Herein, a detailed experiment procedure of TPP workflow was presented, and 28 directly binding proteins and 53 indirectly binding proteins of TH588 was identified for understanding its anti-proliferative functions.