Cancer invasion and metastasis occur in a complex three-dimensional environment, with reciprocal feedback from the surrounding host tissue and vasculature governing cancer cell behaviour. Intravital (in vivo) imaging is providing new insights on how cells behave in their native microenvironment in real-time, thereby improving our understanding of disease progression. Here, for the first time, we have successfully analysed the spatial and temporal regulation of Src activity in response to the anti-invasive Src inhibitor dasatinib in a live animal model of pancreatic cancer using a FLIM-FRET Src-biosensor to monitor drug targeting efficacy. We show that in contrast to conventional techniques, FLIM-FRET analysis allows for accurate, time-dependent, live monitoring of drug efficacy and clearance in vivo. In three-dimensional organotypic assays we demonstrate that a spatially distinct gradient of Src activity exists within invading tumour cells, governed by the depth of penetration into complex matrices. In line with this, we show that a gradient of Src activity also exists within the local microenvironment in live tumours in vivo, where Src activity is enhanced at the invasive border relative to the tumour cortex. Upon treatment with dasatinib we observe a switch in activity at the invasive border from a predominantly active to inactive form, correlating with their impaired metastatic capacity in vivo. Moreover, the regulation of Src in vivo is governed by the proximity of cells to the host vasculature as cells distal to the vasculature are differentially regulated in response to drug treatment compared to cells proximal to vasculature. This suggests that in live tumours a threshold of drug penetrance exists and could potentially be used to map areas of drug targeting efficiency within specific tumour microenvironments. Employment of FLIM-FRET in this capacity can therefore be used as a pre-clinical tool for evaluation of treatment responses in animal models prior to clinical investigations.