Nanoscale carriers with an acid-labile linker between the carrier and drug are commonly used for drug delivery. However, their efficacy is potentially limited by inefficient linker cleavage, and lysosomal entrapment of drugs. To address these critical issues, we developed a new imaging method that spatially overlays the location of a nanoparticle and the released drug from the nanoparticle, on a map of the local intracellular pH that delineates individual endosomes and lysosomes, and the therapeutic intracellular target of the drug—the nucleus. We used this method to quantitatively map the intracellular fate of micelles of a recombinant polypeptide conjugated with doxorubicin via an acid-labile hydrazone linker as a function of local pH and time within live cells. We found that hydrolysis of the acid-labile linker is incomplete because the pH range of 4–7 in the endosomes and lysosomes does not provide complete cleavage of the drug from the nanoparticle, but that once cleaved, the drug escapes the acidic endo-lysosomal compartment into the cytosol and traffics to its therapeutic destination—the nucleus. This study also demonstrated that unlike free drug, which enters the cytosol directly through the cell membrane and then traffics into the nucleus, the nanoparticle-loaded drug almost exclusively traffics into endosomes and lysosomes upon intracellular uptake, and only reaches the nucleus after acid-triggered drug release in the endo-lysosomes. This methodology provides a better and more quantitative understanding of the intracellular behavior of drug-loaded nanoparticles, and provides insights for the design of the next-generation of nanoscale drug delivery systems.