Many tumor-associated mutations result in the abnormal regulation of protein kinases involved in the progression throughout the cell division cycle. The cyclin-dependent kinase (CDK) family has received special attention due to their function as sensors of the mitogenic signals and their central role in cell proliferation. These kinases are frequently upregulated in human cancer most frequently due to overexpression of their cyclin partners or inactivation of the CDK inhibitors. A plethora of small-molecule CDK inhibitors have been characterized in the last years and some of them are currently under clinical development. Other serine-threonine protein kinases such as the Aurora proteins (mostly Aurora A and B) or Polo-like kinases (PLK1) are receiving increased attention as putative cancer targets. Other less studied mitotic kinases such TTK (MPS1), BUB and NEK proteins might also be relevant candidates as new targets of interest in cancer therapy since they play relevant roles on mitotic progression and the spindle checkpoint. Although targeting cell cycle kinases is an efficient procedure to arrest cell proliferation, the best strategy to potently and specifically inhibit tumor cell proliferation is not obvious yet. Thus, some cell cycle kinases may be of interest as targets to abrogate checkpoints and favor apoptotic cell death in tumor cells. New biochemical and genetic studies are required to clarify the use of these kinases as targets in new opportunities to improve cancer therapy.