Successful cancer treatment continues to elude modern medicine and its arsenal of therapeutic strategies. Therapy resistance is driven by tumor heterogeneity, complex interactions between malignant, microenvironmental and immune cells and signaling pathway cross talk. With the ultimate goal of improved molecularly targeted therapeutic efficacy, we have developed and optimized a fluorescence imaging platform termed TRIPODD (Therapeutic Response Imaging through Proteomic and Optical Drug Distribution), resulting in the only methodology capable of simultaneous quantification of single-cell drug target availability and protein expression with preserved spatial context within a tumor. TRIPODD will enable an improved mechanistic understanding of clinically-relevant treatment regimens through spatially resolved single-cell quantification of drug concentration and proteomic response to identify mechanisms of resistant subclonal population outgrowths driving therapeutic resistance.