Development of sensitive and selective strategies for detection of disease biomarkers constitutes a major challenge for diagnostics and companion assays. New strategies are required to advance personalized and point-of-care medicine. Protein kinases (PK) are hyperactivated in many human cancers thereby constituting relevant biomarkers and attractive pharmacological targets.Yet quantification of PK expression levels alone does not convey an accurate readout of their activity, since PKs are subject to numerous interactions and post-translational regulations. Hence technologies that report on PK function are essential for complete appreciation of their behaviour and dysregulation in physio-pathological settings. This review focuses on fluorescent peptide biosensors designed to report on PK activities, and in particular on a toolbox of biosensors tailored to monitor cyclin-dependent kinases. These selective optical probes offer a straightforward means of quantifying differences in kinase activities between healthy and cancer cells, and can report on alterations in response to therapeutics in vitro, in living cells and in vivo.