Chromosome segregation is under strict control of the spindle assembly checkpoint (SAC). The SAC regulates anaphase-promoting complex/cyclosome (APC/C)-dependent proteolysis of securin and cyclin B. Unattached or misaligned chromosomes trigger SAC-mediated mitotic delay by stabilizing securin and cyclin B due to inhibition of APC/C until the problem is solved. Here we present a hitherto unavailable model facilitating the simultaneous depiction of chromosome movements and pulse-chased cyclin B proteolysis in every single cell within a cell population. During chromosome misalignment, we observed slow cyclin B degradation, which changed to fast degradation once the SAC was satisfied, initiating chromosome separation and mitotic exit. Slow degradation during a SAC-mediated mitotic delay is part of a tightly regulated balance between cyclin B synthesis and degradation. Since chromosomal misalignment is a rare event, the ability to study entire cell populations enabled us to monitor for the first time SAC surveillance in living cells without the need of highly artificial perturbation by spindle poisons.