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 cross talk between signaling pathways. Advances in molecular characterization technologies such as next generation sequencing have helped unravel this interaction network and identify therapeutic targets. Tyrosine kinase inhibitors (TKI) are a class of molecularly targeted therapeutics seeking to inhibit signaling pathways critical to sustaining prolifierative signaling, resisting cell death, and the other hallmarks of cancer. While tumors may initially respond to TKI therapy, disease progression is inevitable due to mechanisms of acquired resistance largely involving cellular signaling pathway reprogramming. With the ultimate goal of improved molecularly targeted therapeutic efficacy, our group has developed intracellular paired agent imaging (iPAI) to quantify drug target intereactions and oligonucleotide conjugated antibody (Ab-oligo) cyclic immunofluorescence (cycIF) imaging to characterize perturbed signaling pathways in response to therapy.