The activity of cyclin-dependent kinases (CDKs) lies at the heart of cell cycle and checkpoint control. These complexes have been called the “cell cycle engine,” as it is their enzymatic activity that pushes the cells through the cell-division cycle. If this activity is down-regulated, for example in response to DNA damage and the activation of checkpoint signaling, cell cycle progression is arrested until further notice. In the laboratory, the enzymatic activity of the various CDK complexes can be determined by an in vitro kinase activity assay. In this assay, individual CDK complexes are isolated with the use of specific antibodies and subsequently incubated with a protein substrate in the presence of radioactive ATP. After the termination of this reaction, the amount of radioactively labeled substrate, which reflects the extent of kinase activity, can be visualized by running it on a polyacrylamide gel and exposing it to film. Here, I describe the execution of this in vitro kinase activity assay as well as appropriate controls that need to be considered for the proper evaluation of the results.