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Current clinical methods for the separation of whole blood into blood cells and cell-free plasma are currently based on large facility equipment. The disadvantage of this process is that the patients must have assays performed at the hospital where the separation facility is located. The present study presents a design for microfluidic chips with different microchannel structures, which utilizes backward facing step geometry and centrifugal force to extract the cell-free plasma from whole blood samples at the branch of the microchannel for further assay, avoiding the influence of blood cells. Numerical simulation was performed on a personal computer to analyze the effects of inlet velocity and the structures of the microchannel on the flow field and back-flow in the microchannel, as well as the efficiency of separation and the volumetric fraction of the flowrate of plasma extraction. Based on the numerical simulations, the design with both converging and bending channels was the best design among the two layouts proposed. The experimental results agreed with the numerical predictions well in the present study.