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Accurate, unambiguous detection of molecular interactions in living cells via measurements of Förster (or fluorescence) resonance energy transfer (FRET) events is experimentally challenging. We develop and apply a physiological fluorescence lifetime imaging microscopy (physiological FLIM) system to significantly improve FRET detection in living cells. Multiple positive and negative cellular controls are implemented to validate the experimental method developed. FLIM measurement techniques were found to remove fluorescence intensity-based artifacts, resulting in a seven-fold improvement in fluorescence measurement precision. The addition of cellular environmental controls, including both temperature and stabilization, for physiological FLIM eliminates nonspecific FRET in the live-cell system studied. Overall, only physiological FLIM results in statistically significant results that clearly indicated the …