A microfluidic system was developed for blood plasma separation at high flow rate. This system uses only hydrodynamic forces to separate plasma from whole blood. The microfluidic network features a series of constrictions and bifurcations to enhance the product yield and purity. A maximum purity efficiency of 100% is obtained on blood with entrance hematocrit level up to 30% with a flow rate of 2 mL h-1. Flow cytometry was performed on the extracted plasma to evaluate the separation efficiency and to assess cell damage. A core target of this study was the detection of cell-free DNA from the on-chip extracted plasma. To this effect, PCR was successfully carried out off-chip on the cell-free DNA present in the plasma extracted on-chip. A house-keeping gene sequence (GAPDH) was amplified without the need for a purification after the separation, thereby showing the high quality of the plasma sample. The resulting data suggests that the system can be used as a preliminary module of a total analysis system for cell-free DNA detection in human plasma. © 2010 The Royal Society of Chemistry.