This chapter describes the technology of free flow electrophoresis (FFE) and protocols to separate human plasma for proteome analysis. FFE is a highly versatile technology applied in the field of proteomics because of its continuous processing of sample and high resolution in separation of most kinds of charged or chargeable particles including ions, proteins peptides, organelles, and whole cells. FFE is carried out in an aqueous medium without inducing any solid matrix, such as acrylamide, so that it simplifies complex sample for the downstream analysis. Two FFE protocols are described to separate human plasma proteins under native and denaturing conditions. Plasma separated under native conditions was pooled into acidic-, alkaline-, and albumin- fractions that were furthered for gel-based analysis. Under denaturing condition plasma proteins were separated into 96 fractions. Each fraction can be supplied for in-solution digestion and further LC-MS/MS analysis. From a single FFE fraction 46 different proteins (protein family) have been identified, demonstrating FFE as a high efficient separation tool for human plasma proteome studies.