Circulating tumor cells (CTCs) experience hemodynamic shear stress in circulation and play critical roles in cancer metastasis. The effect of shear on CTCs, however, remains less studied. Here, we described a protocol to circulate HCT116 human colon cancer cells in a microfluidic circulatory system mimicking physiologically relevant circulating conditions. This protocol represents a useful scaffold to mimic the transportation of CTCs in circulation and thus provides an effective means to study the effect of shear on CTCs. We anticipate that future studies using the developed system will help us to further investigate the regulatory roles of shear in molecular responses of CTCs.