A facile and general synthetic methodology has been developed to prepare high drug loading heterotelechelic polymer prodrug nanoparticles; that are nanoparticles obtained by formulation of linear polymers bearing two different molecules at both extremities (at least one of them being a drug). The method relied on the combination of the “drug-initiated” method to obtain α-functional polymer prodrugs by nitroxide-mediated polymerization, followed by the nitroxide exchange reaction from a functional nitroxide to install another molecule of interest at the ω chain-end. To validate the proof of concept, two different combinations have been synthesized using polyisoprene (PI) as the polymer building block: one based on gemcitabine and rhodamine (Gem-PI-Rho) for drug delivery and imaging purposes, and one with aminoglutethimide and doxorubicin (Agm-PI-Dox) for combination therapy purposes. Fluorescent polymer prodrug nanoparticles (Dz = 161 nm) were easily obtained by co-nanoprecipitation of Gem-PI and 5 wt% of Gem-PI-Rho. They were observed by confocal microscopy and they also induced significant cytotoxicity on the human breast cancer cell line MCF-7. As for combination therapy purposes, Agm-PI-Dox nanoparticles (Dz = 63–122 nm) led to enhanced cytotoxicity against MCF-7 cells compared to both monofunctional polymer prodrug nanoparticles (ca. 50% decrease in IC50 vs. Agm-PI and 65% decrease in IC50 vs. PI-Dox) or their equimolar co-nanoprecipitation. This simple and general methodology may pave the way to the design of a broad range of different heterotelechelic polymer prodrug nanoparticles.