Subcellular imaging of tissue in living animals has become increasingly important for studying the dynamic aspects of cancer initiation, progression, invasion and chemoresistance. Intravital imaging of these processes holds promise for discovering novel therapeutic targets of this disease. Here, we assess the role of the multifunctional protein Focal Adhesion Kinase (FAK) in the context of pancreatic cancer, and describe the protocol for surgical implantation of an abdominal imaging window, which will enable visualisation of FAK activity in vivo at a subcellular resolution. FAK is often overexpressed or activated in cancers, such as pancreatic cancer, and is inversely correlated with patient prognosis. Using a primary pancreatic cancer cell line derived from a mouse model of invasive and metastatic pancreatic ductal adenocarcinoma, in combination with the FAK inhibitor PF-562271, we show that FAK inhibition alters migratory and adhesion patterns on extracellular matrix mimics, reduces cell invasion and survival, and reduces cell growth upon loss of attachment. The rapid resistance of pancreatic cancer to current chemotherapeutics led to the assessment of PF-562271 as a potential sensitiser to the current standard-of-care treatments, Gemcitabine and Abraxane. Using second harmonic generation imaging, we show that PF-562271 mediated FAK inhibition suppresses fibroblast-driven ECM remodelling, which may prove beneficial in overcoming the fibrotic pancreatic cancer stroma. Finally, we have generated a stable cell line expressing the FAK-Fluorescent Resonance Energy Transfer (FRET) biosensor, which we plan to couple with our state-of-the-art abdominal imaging window technique, to provide an accurate readout of the dynamic spatiotemporal activity of FAK in vivo.