Nanoformulations have shown great promise for delivering chemotherapeutics and hold tremendous clinical relevance. However nuclear mapping of the chemodrugs is important to predict the success of the nanoformulation. In this study fluorescence microscopy and a subcellular tracking algorithm were used to map the diffusion of chemotherapeutic drugs in cancer cells. Positively charged nanoparticles efficiently carried the chemodrug across the cell membrane. The algorithm helped map free drug and drug-loaded nanoparticles, revealing a varying nuclear diffusion pattern of the chemotherapeutics in drug-sensitive and -resistant cells in a live dynamic cellular environment. While the drug-sensitive cells showed an exponential uptake of the drug with time, resistant cells showed random and asymmetric drug distribution. Moreover nanoparticles carrying the drug remained in the perinuclear region, while the drug accumulated in the cell nuclei. The tracking approach has enabled us to predict the therapeutic success of different nanoscale formulations of doxorubicin. 漏 This article not subject to U.S. Copyright. Published 2012 by the American Chemical Society.