Indoleamine 2,3-dioxygenase 1 (IDO1) is a potently immunosuppressive protein that inhibits antitumor immunity through both tryptophan metabolism and non-enzymatic functions. Pharmacological therapies targeting IDO1 enzyme activity have generally failed to improve the overall survival of patients with cancer. Developing new therapeutic agents that are capable of neutralizing both enzyme-and non-enzyme-derived immunosuppressive IDO1 effects is therefore of high interest. We previously described the development of a novel Proteolysis Targeting Chimera (PROTAC), NU223612, that degrades IDO1 in cultured human glioblastoma (GBM) cells, as well as in well-established brain tumors, in vivo . In this study, we rationally optimized the composition, rigidity, and linker orientation of the PROTAC structure to create NU227326, which degrades IDO1 with a DC 50 of 5 nM in human GBM cells. Mechanistic studies showed that IDO1 degradation occurred through the ubiquitin-proteasome system and was sustained for at least 2 days, supporting NU227326 as a highly potent IDO1 PROTAC suitable for further studies in GBM and other human cancers.