Fluorescence Lifetime Förster Resonance Energy Transfer (FLIM-FRET) is a unique non-invasive imaging platform to monitor and quantify in vivo target engagement in pre-clinical studies. FLIM FRET is a valuable tool in targeted drug delivery due to its nanoscale-range molecular resolution that detects near-infrared labeled ligand binding to dimerized receptors followed by their uptake into cancer cells in vivo. Various imaging platforms, including PET, lack the ability to directly discriminate between unbound and internalized ligands. Since transferrin receptor (TfR) level is significantly elevated in cancer cells compared to non-cancerous cells, transferrin (Tf) has been successfully used in molecular imaging and targeted anti-cancer drug delivery. The dimeric nature of TfR allows for the quantification of Tf internalization into cancer cells by measuring FLIM FRET between receptor-bound Tf donor and acceptor NIR fluorophore pairs, based on the reduction of donor fluorophore lifetime in live mice. We analyzed tumor morphology, the level of expression of TfR, estrogen receptor (ER) and Tf accumulation in human breast cancer tumor xenografts. We found a remarkable heterogeneity of breast cancer tumors regarding their size, cell density, TfR and ER expression and Tf uptake. The results of this study confirm a strong correlation between in vivo NIR FLIM FRET and ex vivo evaluation of Tf uptake into tumor tissues, thus validating FD% as a robust measure of the target engagement of TfR-Tf in tumor cells in vivo.