Cell division in mammalian cells is driven by protein kinases that regulate progression through the various phases of the cell cycle. Cyclin-dependent kinases (Cdks) regulate cell cycle commitment, DNA synthesis and the onset of mitosis. Kinases of the Aurora, Polo and Nek families participate in the centrosome cycle and modulate spindle function. Additional kinases such as Bub1, BubR1 and Mps1 regulate the spindle assembly checkpoint. It has been well established that misregulation of Cdks is one of the most frequent alterations in human cancer. Recent evidence indicates that mutations involving mitotic kinases are also linked to tumor development. These findings suggest novel strategies to use cell cycle kinases as targets for therapeutic intervention.