It is beneficial to investigate the effect of nanomedicines on cancer cell behaviors on three-dimensional (3D) platforms for evaluating and developing novel antitumor nanomedicines in vitro. Two-dimensional flat surface is widely used to study the cytotoxicity of nanomedicines on cancer cells, and there is little work using 3D confinement to assess the effect of nanomedicine on cancer cells. In this work, PEGylated paclitaxel nanoparticles (PEG-PTX NPs) were applied for the first time to treat nasopharyngeal carcinoma (NPC43) cells in 3D confinement which consisted of microwells with different sizes and a glass cover. The cytotoxicity of the small molecule drug paclitaxel (PTX) and PEG-PTX NPs were studied in microwells with sizes of 50×50, 100×100, and 150×150 μm2 and with covers. After NPC43 cells were treated with PTX and PEG-PTX NPs, the cell migration speed and cell morphology were analyzed in different microwells without and with covers. The relationship between the cytotoxicity from drugs, the size effect from microwells, and cell behaviors were analyzed. These results not only show the effect of 3D confinement on cytotoxicity of nanomedicines and cell behaviors, but also provide a novel method to screen anticancer drugs and evaluate the cell behaviors in vitro.