Signaling networks that control cell behavior are tightly regulated in spac and time. Fluorescent biosensors for living cells can provided a valuable window on the dynamics of these networks, providing quantitative information on the kinetics and localization of protein activity in vivo. However, biosensors for living cells currently require considerable optimization for each target and are also limited by the availability of naturally occurring ligands/binding elements with appropriate target specificity. In this talk, I will describe a new approach of generating biosensors based on an engineered fibronectin monobody scaffold that can be tailored to bind different targets via high throughput screening. Using the artificial monobody scaffold and extremely bright reporter fluorescent dyes, we generated a biosensor that can report the activation of endogenous, unmodified Src family kinases (SFK) in living cells. The new SFK biosensor in conjunction with automated analysis of cell edge dynamics provides revealing insights into the role of Src kinases in cell migration. I will discuss my future plans regarding generating biosensors for individual Src family kinases that are important in cancer and other diseases. These new biosensors will help address the underlying plasticity in cell signaling, and help study fundamental aspects of cell adhesion and migration. The development of a new class of biosensors based on small molecules that are specific for active conformations of signaling proteins will be discussed. I will also discuss new ways of probing cell heterogeneity in cancer and stem cells using high throughput, high content imaging.