Doxorubicin (DOX) is a potent anticancer drug used for the treatment of leukemia, lymphomas, and other solid tumors. To enhance specific and reduce unfavorable side effects of DOX, the use of polymeric carriers has been frequently employed. Our laboratory has developed a Gelatin DOX conjugate (GDOX) to improve treatment efficacy for breast cancer. The overall goal of this research is to study the intracellular trafficking of GDOX in Human Caucasian breast adenocarcinoma (MCF-7) cells. DOX is a highly fluorescent compound, with an excitation/emission wavelength of 470/595 nm. This allows visualization of the cellular internalization and distribution of the DOX/GDOX using fluorescence microscopy techniques. In the first chapter, a procedure for the live-cell imaging of DOX/GDOX in MCF-7 was developed. In the second chapter, a surrogate conjugate (GDye) was synthesized with no DOX attached, to act as a marker for GDOX. Characterization of the GDye was performed by high-performance size exclusion chromatography (HPSEC). Intracellular trafficking studies with GDye showed no noticeable effect of gelatin molecular weight distribution. Two dye loads were used to study the kinetics of uptake in MCF-7 cells. Finally, the release of free DOX from GDOX was demonstrated by quantitating its escape from lysosomes and accumulation in the nucleus of MCF-7 cells. Our results showed that the nuclear accumulation of DOX released from the GDOX was 11-fold lower than that of DOX alone, thereby suggesting the possibility for GDOX toxicity via a nuclear pathway.