Cell cycle checkpoints are surveillance mechanisms that sequentially and 
continuously monitor cell cycle progression thereby contributing to the 
preservation of genetic stability. Among them, the spindle assembly checkpoint 
(SAC) prevents the occurrence of abnormal divisions by halting the metaphase to 
anaphase transition following the detection of erroneous 
microtubules-kinetochore attachment(s). Most synchronization strategies are 
based on the activation of cell cycle checkpoints to enrich the population of 
cells in a specific phase of the cell cycle. Here, we develop a two-step 
protocol of sequential cell synchronization and desynchronization employing 
antimitotic SAC-inducing agents (i.e., nocodazole or paclitaxel) in combination 
with the depletion of the SAC kinase MPS1. We describe cytofluorometric and 
time-lapse videomicroscopy methods to detect cell cycle progression, including 
the assessment of cell cycle distribution, quantification of mitotic cell 
fraction, and analysis of single cell fate profile of living cells. We applied 
these methods to validate the synchronization-desynchronization protocol and to 
qualitatively and quantitatively determine the impact of SAC inactivation on the 
activity of antimitotic agents.