Hemolysis, involving the rupture of red blood cells (RBCs) and release of their contents into blood plasma, is a major issue of concern in clinical fields. Hemolysis in vitro can occur as a result of errors in clinical trials; in vivo, hemolysis can be caused by a variety of medical conditions. Blood plasma separation is often the first step in blood-based clinical diagnostic procedures. However, inhibitors released from RBCs due to hemolysis during plasma separation can lead to problems in diagnostic tests such as low sensitivity, selectivity and inaccurate results. In particular, a general lack of simple and reliable blood plasma separation methods has been a major obstacle for microfluidic-based point-of-care (POC) diagnostic devices. Here we present a hemolysis-free microfluidic blood plasma separation platform. A membrane filter was positioned on top of a vertical up-flow channel (filter-in-top configuration) to reduce clogging of RBCs by gravity-assisted cells sedimentation. With this device, separated plasma volume was increased approximately 4-fold (2.4 μL plasma after 20 min with 38% hematocrit human whole blood), and hemoglobin concentration in separated plasma was decreased approximately 90% due to the prevention of RBCs hemolysis, when compared to conventional filter-in-bottom configuration blood plasma separation platforms. On-chip plasma contained ~90% of protein and ~100% of nucleic acids found in off-chip centrifuged plasma, confirming comparable target molecule recovery efficiency. This simple and robust on-chip blood plasma separation device integrates with downstream detection modules to ultimately create sample-to-answer microfluidic POC diagnostics devices.