Microtubule-targeting agents (MTAs) have been used for decades to treat different hematologic and solid cancers. The mode of action of these drugs mainly relies on their ability to bind tubulin subunits and/or microtubules and interfere with microtubule dynamics. In addition to its MTH1-inhibiting activity, TH588 has been recently identified as an MTA, whose anticancer properties were shown to largely depend on its microtubule-targeting ability. Although TH588 inhibited tubulin polymerization in vitro and reduced microtubule plus-end mobility in interphase cells, its effect on microtubule dynamics within the mitotic spindle of dividing cells remained unknown. Here, we performed an in-depth analysis of the impact of TH588 on spindle-associated microtubules and compared it to the effect of low-dose nocodazole. We show that both treatments reduce microtubule turnover within the mitotic spindle. This microtubule-stabilizing effect leads to premature formation of kinetochore-microtubule end-on attachments on uncongressed chromosomes, which consequently cannot be transported to the cell equator, thereby delaying cell division and leading to cell death or division with uncongressed chromosomes.