Exposure to drugs that interfere with microtubule dynamics block cell cycle progression at mitosis by prolonged activation of the spindle assembly checkpoint (SAC). Cells can evade mitotic arrest and proceed to interphase without chromosome segregation by a process termed mitotic slippage that involves Cyclin B1 degradation without checkpoint inactivation. Here, we explored the cellular response to small-molecule inhibitors of Polo-like kinase 1 (Plk1), an important regulator of cell division. We found that the clinical Plk1 inhibitors BI 2536 and BI 6727, both unexpectedly, induced a dose-dependent cellular drug response: While mitotic arrest was induced in cancer cell lines and primary non-transformed cells across the entire range of concentrations tested, only high concentrations seemed to promote mitotic slippage. Since this observation contrasts with the effects expected from studies reporting RNAi-mediated Plk1 depletion in cancer cells, we wondered whether both ATP-competitive inhibitors target unknown kinases that are involved in signaling from the spindle assembly checkpoint (SAC) and might contribute to the mitotic slippage. A chemical proteomics approach used to profile the selectivity of both inhibitors revealed that SAC kinases are not targeted directly. Still, the activities of Cdk1/Cyclin B1 and Aurora B, which plays important roles in the error correction of false microtubule-kinetochore attachments and in checkpoint signaling, were shown to be downregulated at high inhibitor concentrations. Our data suggest that the inhibition of Plk1 activity below a certain threshold influences Aurora B activity via reduced phosphorylation of Fox M1 and Survivin leading to diminished levels of Aurora B protein and alteration of its subcellular localization. Within the spectrum of SAC proteins that are degraded during mitotic slippage, the degradation of Cyclin B1 and the downregulation of Aurora B activity by Plk1 inhibition seem to be critical promoters of mitotic slippage. The results indicate that careful dose-finding studies in cancer trials are necessary to limit or even prevent mitotic slippage, which could be associated with improved cancer cell survival.