The mitotic shake-off method is a laboratory technique used to isolate cells that are actively undergoing mitosis from a population of cells in culture. During mitosis, cells undergo a series of characteristic changes. After the cells have been induced to enter mitosis, they are gently dislodged from the culture vessel using a mild mechanical agitation technique, such as shaking or tapping. Since cells in mitosis typically round up and detach from the substrate as they prepare to divide, they are more easily released from the culture surface compared to cells in other phases of the cell cycle. Those isolated mitotic cells can be transferred to other cell culture vessels to release cell cycle progression. Mitotic cells go through next cell cycle phases with time. Using this method, cell cycle-dependent cellular activity such as cell cycle-specific protein expression and activation, and cell cycle-specific sensitivity to agents, can be carried out. Since this method is completely drug free, it has series of benefits including no drug cost and no potential toxicity from drug exposure. This chapter introduces cellular synchronization technique by mitotic shake-off method.