Threonine tyrosine kinase (TTK) is a critical component of the spindle assembly checkpoint and plays a pivotal role in mitosis. TTK has been identified as a potential therapeutic target for human cancers. Here, we describe our design, synthesis and evaluation of a class of covalent TTK inhibitors, exemplified by 16 (SYL1073). Compound 16 potently inhibits TTK kinase with an IC of 0.016 μM and displays improved selectivity in a panel of kinases. Mass spectrometry analysis reveals that 16 covalently binds to the C604 cysteine residue in the hinge region of the TTK kinase domain. Furthermore, 16 achieves strong potency in inhibiting the growth of various human cancer cell lines, outperforming its relative reversible inhibitor, and eliciting robust downstream effects. Taken together, compound 16 provides a valuable lead compound for further optimization toward the development of drug for treatment of human cancers.