Centrosome is an evolutionarily conserved organelle that comprises two barrel-shaped centrioles surrounded by pericentriolar material (PCM). It functions as the major microtubule-organizing center (MTOC) to regulate cell polarity, motility, intracellular material transport during interphase, and bipolar spindle assembly during mitosis. Cartwheel assembly is considered the first step in the initiation of procentriole biogenesis at early S phase. In human cells, cartwheel is a transient scaffolding structure that is disassembled during procentriole maturation at mitotic exit. This chapter describes methods for synchronizing mammalian cells into mitosis by small molecule inhibitor treatment and visualizing cartwheel disassembly process during mitosis in fixed and live cells by fluorescence microscope.