Biomaterials in the nanometer size range can be engineered for site-specific delivery of drugs after injection into the blood circulation. However, translation of such nanomedicines from the bench to the bedside is still hindered by many extracellular and intracellular barriers. To realize the concept of targeted drug delivery with nanomedicines, research groups are studying intensively the extra- and intra-cellular mechanisms involved as a response to the physicochemical properties of the nanomedicines. In this review, we highlight the contributions of fluorescence fluctuations spectroscopy techniques to better understand, and in turn to bypass, the major hurdles to therapeutic delivery, focusing mostly on the intracellular dynamics of drug-delivery systems.