We design, develop and demonstrate the principle of a continuous, non-intrusive, low power microfluidics-based lab-ona- chip (LOC) structure for Circulating Tumor Cell (CTC) separation. Cell separation is achieved through 80 cascaded contraction and expansion microchannels of widths 60 μm and 300 μm, respectively, and depth 60 μm, which enable momentum-change-induced inertial forces to be exerted on the cells, thus routing them to desired destinations. The total length of the developed LOC is 72 mm. The LOC structure is simulated using the COMSOL multiphysics software, which enables the optimization of the dimensions of the various components of the LOC structure, namely the three inlets, three filters, three contraction and expansion microchannel segments and five outlets. Simulation results show that the LOC can isolate CTCs of sizes ranging from 15 to 30 μm with a recovery rate in excess of 90%. Fluorescent microparticles of two different sizes (5 μm and 15 μm), emulating blood and CTC cells, respectively, are used to demonstrate the principle of the developed LOC. A mixture of these microparticles is injected into the primary LOC inlet via an electronically-controlled syringe pump, and the large-size particles are routed to the primary LOC outlet through the contraction and expansion microchannels. Experimental results demonstrate the ability of the developed LOC to isolate particles by size exclusion with an accuracy of 80%. Ongoing research is focusing on the LOC design improvement for better separation efficiency and testing of biological samples for isolation of CTCs.