We have characterized several receptor‐probe interactions, such as transferrin‐transferrin receptor,[14, 16, 20, 33, 34] Cetuximab‐Epidermal Growth Factor Receptor,[22] and TZM‐HER2,[21, 35] providing an accurate measurement of bound and unbound fraction of the probe to its respective target.[16, 21, 22] Herein, we present dark quencher‐based MFLI FRET as a unique noninvasive approach to measure the TZM‐HER2 drug‐target engagement of HER2‐positive human breast and ovarian tumor xenografts mice model, which display varying TMEs. This evidence concerns the gene ERBB2 and ovarian neoplasm.