Density gradient centrifugation exploits density differences between different blood cells to accomplish separation of peripheral blood mononuclear cells (PBMCs) from polymorphonuclear (PNM) cells, and erythrocytes or red blood cells (RBCs). While density gradient centrifugation offers a label-free alternative avoiding the use of harsh lysis buffers for blood cell isolation, it is a time-consuming and labor-intensive process during which blood cells are subject to high-levels of centrifugal force that can artifactually activate cells. To provide a low-stress alternative to this elegant method, we miniaturized and automated this process using microfluidics to ensure continuous PBMCs isolation from whole blood while avoiding the exposure to high-levels of centrifugal stress in a simple flow-through format. Within this device, a density gradient is established by exploiting laminar flow within microfluidic channels to layer a thin stream of blood over a larger stream of Ficoll. Using this approach we demonstrate successful isolation of PBMCs from whole blood with preservation of monocytes and different lymphocyte subpopulations similar to that seen with conventional density gradient centrifugation. Evaluation of activation status of PBMCs isolated using this technique shows that our approach achieves minimal isolation process induced activation of cells in comparison to conventional lysis or density gradient centrifugation. This simple, automated microfluidic density gradient centrifugation technique can potentially serve as tool for rapid and activation-free technique for isolation of PBMCs from whole blood for point-of-care applications.