Isolation and processing blood into leukocyte subsets are important processes in research. Although methods have been developed to fractionate small volumes of blood, optimizing the methods and balancing the underlying costs are often necessary. The need for such optimization is particularly critical when processing larger volumes of blood. We describe a simple and reproducible method for processing larger volumes of fresh blood rapidly and consistently, which yields peripheral blood mononuclear cells (PBMCs) and leukocyte subsets with high purity (81-96%; n=13) and higher yields relative to stored blood. RNA isolated from these cells was found to be suitable for downstream applications. Blood stored for 24 hours (n=4) before processing resulted in significantly lower yields of PBMCs (58 percent lower), T cells (52 percent lower), B cells (21 percent lower) and monocytes (25 percent lower) compared to fresh blood. However, the purity of the fractionated cells was comparable to that obtained with fresh blood. Furthermore, we report that the yield and purity of the leukocyte subsets isolated from cryopreserved PBMCs (n=4) were not compromised.