Due to recent advances in nanotechnology, the application of nanoparticles (NPs) in cancer therapy has become a leading area in cancer research. Despite the importance of cancer-associated fibroblasts (CAFs) in creating an optimal niche for tumor cells to grow extensively, most of the work has been focused on tumor cells. Therefore, to effectively use NPs for therapeutic purposes, it is important to elucidate the extent of NP uptake and retention in tumor cells and CAFs. Three tumor cell lines and three CAF cell lines were studied using gold NPs (GNPs) as a model NP system. We found a seven-fold increase in NP uptake in CAFs compared to tumor cells. The retention percentage of NPs was three-fold higher in tumor cells as compared to CAFs. Furthermore, NP uptake and retention were significantly enhanced using a 50 nM concentration of docetaxel (DTX). NP uptake was improved by a factor of three in tumor cells and a factor of two in CAFs, while the retention of NPs was two-fold higher in tumor cells compared to CAFs, 72 h post-treatment with DTX. However, the quantity of NPs in CAFs was still three-fold higher compared to tumor cells. Our quantitative data were supported by qualitative imaging data. We believe that targeting of NPs in the presence of DTX is a very promising approach to accumulate a higher percentage of NPs and maintain a longer retention in both tumor cells and CAFs for achieving the full therapeutic potential of cancer nanotechnology.