This thesis presents a microfluidic magnetophoresis device consisting of a trapezoidal channel containing five side outlet branches and a narrow rectangular channel with three outlet branches. This unique structure enabled the sequential separation of cells loaded with tiny amounts of iron oxide and cells heavily labeled with iron oxide, in a single device. Since accurate assessment potential nanotoxicity on the basis of cellular iron content is of crucial importance, the reliable microfluidic device is required for the precise separation of cells into subpopulations according to their magnetic nanoparticle loading. As a proof of the demonstration of the proposed magnetophoretic platform, we attempted the sequential separation of Raw 264.7 cells with high heterogeneity in uptake capabilities (1-50 pg of iron per cell). Consequently, we were able to differentiate the bulk cell population into seven subpopulations according to their mean iron oxide loading. Before the post-analysis, a simple retrieval method for precipitated particles is applied to prevent sample loss. Finally, We evaluated potential nanotoxicity effects from the production of excess reactive oxygen species (ROS) and the inhibition of proliferation on the separated subpopulations, and found that 46.6% of cells loaded with iron over the threshold value (16.4 pg) had higher ROS levels than the control group. Cells loaded with over 3.7 pg of iron exhibited transiently inhibited cell cycle progression. Furthermore, cells loaded with over 35.4 pg of iron exerted a significant effect on cell proliferation. The proposed system could be useful in the investigation of nanotoxicity effects of iron oxide nanoparticle-induced cells, based on their iron oxide nanoparticle loading.