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Fluorescence spectroscopy, coupled with microscopy, opens new frontiers for the study of dynamic processes with high spatio-temporal resolution. The application of phasor plots to FLIM and hyperspectral imaging demonstrate unprecedented capabilities to study complex photophysics at the subcellular level. Using these approaches we studied the effects of an H2O2 bolus on NIH-3T3 membranes dynamics monitored by LAURDAN fluorescence. Exposure of NIH-3T3 cells to a bolus of H2O2 modifies the cell membranes and, in particular, the plasma membrane in a complex manner. The LAURDAN results reveal that the peroxide treatment decreases membrane fluidity but surprisingly increases dipolar relaxation around the excited probe. Using the Multidimensional-phasor approach we elucidated the complex photophysics of LAURDAN incorporated into cell membrane after H2O2 exposure. The results indicate the occurrence of LAURDAN fast-diffusion from gel↔ld phases in membranes exposed to a H2O2 bolus. An ad hoc hypothesis is presented to interpret the results in the context of H2O2 oxidative distress/eustress.LAURDAN fluorescence and phasor plot identify a complex effect of H2O2 on NIH-3T3 membranes.A H2O2 bolus modifies the supramolecular organization of the plasma membrane in NIH-3T3 cells.Decreased membrane fluidity but increased dipolar relaxation was found after a H2O2 bolus.MultiD-phasor revealed the occurrence of LAURDAN fast-diffusion during the excited-state from gel→ld phases after a H2O2 bolus.