Purpose <p>Glioblastoma (GBM) is the most common adult primary brain tumor for which new therapeutic strategies are desperately needed. Monopolar spindle 1 (MPS1) is a mitotic kinase that plays a pivotal role in the spindle assembly checkpoint (SAC). GBM appears to be dependent on SAC fidelity, as MPS1 is overexpressed in many GBM patients. Thus, inhibiting MPS1 seems a viable therapeutic strategy to enhance mitotic cell death by attenuating SAC fidelity. NTRC 0066-0 is an MPS1 inhibitor that combines low nanomolar potency with a relatively long on-target residence time.</p> Methods <p>We here investigate the potential of NTRC 0066-0 as monotherapy and in combination with chemo-radiation for treatment of GBM using various in vitro and orthotopic in vivo models.</p> Results <p>We show that NTRC 0066-0 efficiently induces GBM cell death in vitro, following continuous exposure with IC₅₀s in the low nanomolar range. In vivo, we demonstrate that NTRC 0066-0 has a high brain penetration, although it is a substrate of the efflux transporter P-glycoprotein at the blood-brain barrier. However, despite using recipient <i>Abcb1a/b; Abcg2</i>^{<i>−/−</i>} mice with superior brain penetration and administering NTRC 0066-0 using a dose-dense regimen, we did not observe antitumor efficacy against an orthotopic GBM mouse model, neither as monotherapy nor in combination with standard-of-care temozolomide chemotherapy and radiotherapy.</p> Conclusion <p>These data indicate that developing MPS1 inhibitors for treatment of GBM will be challenging and would require further understanding of in vivo determinants of translating SAC inhibition to antitumor efficacy.</p>