Herein, we report the direct measurement of TZM–HER2 binding in HER2-positive human breast cancer cells and tumor xenografts using fluorescence lifetime Forster Resonance Energy Transfer (FLI-FRET) via near-infrared (NIR) microscopy (FLIM-FRET) as well as macroscopy (MFLI-FRET) approaches. Here, ERBB2 is linked to neoplasm.