Fluorescence labelling is often used for tracking nanoparticles, providing a convenient assay for monitoring nanoparticle drug delivery. However, it is difficult to be quantitative, as many factors affect the fluorescence intensity. Förster resonance energy transfer (FRET), taking advantage of the energy transfer from a donor fluorophore to an acceptor fluorophore, provides a distance ruler to probe NP drug delivery. This article provides a review of different FRET approaches for the ratiometric monitoring of the self-assembly and formation of nanoparticles, their in vivo fate, integrity and drug release. We anticipate that the fundamental understanding gained from these ratiometric studies will offer new insights into the design of new nanoparticles with improved and better-controlled properties.