Mitosis is the key event of the cell cycle during which the sister chromatids are segregated onto two daughter cells. It is well established that abrogation of the normal mitotic progression is a highly efficient concept for anti-cancer treatment. In fact, various drugs that target microtubules and thus interfere with the function of the mitotic spindle are in clinical use for the treatment of various human malignancies for many years. However, since microtubule inhibitors not only target proliferating cells severe side effects limit their use. Therefore, the identification of novel mitotic drug targets other than microtubules have gained recently much attention. This review will summarize the latest developments on the identification and clinical evaluation of novel mitotic drug targets and will introduce novel concepts for chemotherapy that are based on recent progress in our understanding how mitotic progression is regulated and how anti-mitotic drugs induce tumor cell death.