Cyclin-dependent kinases play central roles in regulation of cell cycle progression, transcriptional regulation and other major biological processes such as neuronal differentiation and metabolism. These kinases are hyperactivated in most human cancers and constitute attractive pharmacological targets. A large number of ATP-competitive inhibitors of CDKs have been identified from natural substances, in high throughput screening assays, or through structure-guided approaches. Alternative strategies have been explored to target essential protein/protein interfaces and screen for allosteric inhibitors that trap inactive intermediates or prevent conformational activation. However this remains a major challenge given the highly conserved structural features of these kinases, and calls for new and alternative screening technologies. Fluorescent biosensors constitute powerful tools for the detection of biomolecules in complex biological samples, and are well suited to study dynamic processes and highlight molecular alterations associated with pathological disorders. They further constitute sensitive and selective tools which can be readily implemented to high throughput and high content screens in drug discovery programmes. Our group has developed fluorescent biosensors to probe cyclin-dependent kinases and gain insight into their molecular behaviour in vitro and in living cells. These tools provide a means of monitoring subtle alterations in the abundance and activity of CDK/Cyclins and can respond to compounds that interfere with the conformational dynamics of these kinases. In this review we discuss the different strategies which have been devised to target CDK/Cyclins, and describe the implementation of our CDK/Cyclin biosensors to develop HTS/HCS assays in view of identifying new classes of inhibitors for cancer therapeutics.