Abstract: A multiphoton fluorescence anisotropy microscopy live cell imaging system and method to measure and map drug-target interaction in real lime at subcellular resolution. Proposed modality enables a direct measurement of drug/target binding in vivo, high-resolution spatial and temporal snapping of bound and unbound drug distribution, and presents an versatile tool to enhance understanding of drug activity. Application of tire system to measurement of intracellular target engagement of the chemotherapeutic Olaparib, a poly(ADP-ribose) polymerase inhibitor, in live cells and within a tumor in vivo.