The complexity and specificity of many forms of signal transduction are widely suspected to require spatial microcompartmentation and dynamic modulation of the activities of protein kinases, phosphatases and second messengers. However, traditional methodologies for detecting signaling events, such as activation of kinases, second messenger production and degradation, are limited in their spatiotemporal resolution and do not allow following these events within the live-cell context. To achieve dynamic tracking of signaling activities in living cells, genetically encoded fluorescent reporters for protein kinases, second messengers such as cyclic AMP and phosphoinositides have been engineered. Their development and specific examples of their application will be discussed. For instance, this approach has revealed distinct pools of second messengers in specific cellular locations, such as the mitochondria and nucleus. In addition, a novel live-cell high-throughput screening method has been developed for identification of new modulators that affect the dynamic activity of kinases and second messengers.