Analytical time resolved Förster resonance energy transfer (FRET) can be exploited for assessing, in cells and tumor micro arrays, the activation status and dynamics of oncoproteins such as epidermal growth factor receptor (EGFR1) and their downstream effectors such as protein kinase B (PKB) and 3-phosphoinositide-dependent protein kinase 1 (PDK1). The outcome of our research involving the application of quantitative imaging for investigating molecular mechanisms of phosphoinositide-dependant enzymes, such as PKB and PDK1, has resulted in a refined model describing the dynamics and regulation of these two oncoproteins in live cells. Our translational research exploits a quantitative FRET method for establishing the activation status of predictive biomarkers in tumor micro arrays. We developed a two-site FRET assay monitored by automated frequency domain Fluorescence lifetime imaging microscopy (FLIM). As a proof of principle, we tested our methodology by assessing EGFR1 activation status in tumor micro arrays from head and neck patients. Our two-site FRET assay, by high-throughput frequency domain FLIM, has great potential to provide prognostic and perhaps predictive biomarkers.