We demonstrate the use of surface Zeta potential measurements as a new tool to investigate the interactions of iron oxide nanoparticles and cowpea mosaic virus (CPMV) nanoparticles with human normal breast epithelial cells (MCF10A) and cancer breast epithelial cells (MCF7) respectively. A substantial understanding in the interaction of nanoparticles with normal and cancer cells in vitro will enable the capabilities of improving diagnostic and treatment methods in cancer research, such as imaging and targeted drug delivery. A theoretical Zeta potential model is first established to show the effects of binding process and internalization process during the nanoparticle uptake by cells and the possible trends of Zeta potential change is predicted for different cell endocytosis capacities. The corresponding changes of total surface charge of cells in the form of Zeta potential measurements were then reported after incubated respectively with iron oxide nanoparticles and CPMV nanoparticles. As observed, after MCF7 and MCF10A cells were incubated respectively with two types of nanoparticles, the significant differences in their surface charge change indicate the potential role of Zeta potential as a valuable biological signature in studying the cellular interaction of nanoparticles, as well as specific cell functionality.