By applying novel intravital imaging techniques to dynamically monitor pathway activity upon therapeutic inhibition, we are assessing chemoresistance within the hypoxic microenvironment of pancreatic ductal adenocarcinoma (PDAC). PDAC presents one of the most common causes of cancer death in many counties and has proven resistant to currently available therapies. Hypoxia is a negative prognostic factor in PDAC, known to increase radioresistance, chemoresistance, angiogenesis and metastasis. Here we demonstrate pronounced chemoresistance within hypoxia to three clinically relevant inhibitors of the PI3K pathway; Rapamycin (mTORC1 inhibitor), NVP-BEZ235 (dual PI3K/mTORC1 inhibitor) and AZD2014 (dual PI3K/mTORC2 inhibitor). Utilizing novel mouse models of PDAC with p53 loss or gain-of-function mutations in p53, we have expressed an optimized FRET biosensor to reversibly read out Akt activity in real time. In cancer, the three Akt homologs Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ have been implicated in aberrant cell survival, growth, proliferation, angiogenesis, metabolism and migration. Hence, by using Akt activity as our readout of PI3K pathway activity, we can dynamically monitor inhibitory effects within our in vivo mouse models. By targeting multiple levels of the PI3K pathway, using both in vivo mouse models and high fidelity three-dimensional assays, we are unraveling the best targets for both anti-invasive and anti-proliferative effects, in the context of modulating the hypoxic microenvironment.