The tools of molecular imaging can provide spatially- and temporally-resolved information on biological structures and functions. Because investigators have increasingly recognized the importance of context in the study of gene expression and protein function as well as understanding regulatory mechanisms within cellular micro-environments, many have turned to non-invasive imaging technologies to advance research on human health and disease. These non-invasive imaging strategies can interrogate protein processing, protein-protein interactions, gene expression and flux through metabolic pathways in real-time in cells and live animals, and are increasingly useful in understanding signal transduction and pathobiology of human diseases, including cancer, to facilitate development of effective therapies. His group has used these approaches to investigate mechanisms of regulation of IkB/NFkB signaling, b -catenin processing, multidrug resistance, EGFR signaling, and innate immunity. They have developed high fidelity molecular-specific pharmacodynamic reporters of several anti-cancer drugs useful in whole animal models. They have created a widely-used luciferase protein fragment complementation platform, dual-color protein interaction switches, and fusion reporters for bioluminescent analysis of protein processing in living animals, each with broad applications in biotechnology and biomedicine. By making these constructs and methods freely available, the team helped catalyze the application of genetically-encoded molecular imaging approaches to diverse medical and basic biological questions in a variety of fields. On other fronts, Piwnica-Worm’s group has moved several of their scientific discoveries from bench to bedside, facilitating the goals of precision medicine by translating novel PET tracers and cell-penetrating activatable peptides toward clinical imaging applications.