Combinatory anticancer drug release from gold nanoparticles (AuNPs) in K562 human myeloid leukemia cells was performed using Raman spectroscopy. We fabricated the anticancer drug of imatinib as a BCR-ABL tyrosine kinase inhibitor on AuNP surfaces along with a transferrin (Tf)-targeting moiety to treat the leukemia cells. DNA topoisomerase I inhibitor topotecan was also assembled to monitor its fluorescence onto AuNPs. The linker group of 4-carboxylic benzoic acid was used to conjugate to targeting the Tf protein. Our Raman data indicated that the drug molecules were not detached in the cell culture media but released after treatment with glutathione (2 mM). Intracellular distribution and release of the anticancer drug–AuNP conjugates in K562 cells were examined by both fluorescence microscopy and dark-field microscopy with surface-enhanced Raman scattering. Copyright © 2013 John Wiley & Sons, Ltd.