Extracellular vesicles (EV), cell-derived spherical particles enclosed by a lipid-bilayer, contain various molecules and mediate cell-to-cell communication. EV in plasma have been expected as potential disease biomarkers. However, the high concentration of lipoproteins in plasma, which have similarities in size, density, and contents to EV, hampers analysis on plasma EV. To overcome this issue, we aimed to develop an effective isolation method for plasma EV excluding lipoproteins (HDL, LDL/VLDL), by using polyanion and a divalent cation. Human plasma was mixed with 1) phosphotungstic acid and MgCl₂, 2) heparin and MnCl₂, and 3) polyethylene glycol (PEG). The mixture was centrifuged to separate a supernatant and pellet. 4) Dextran sulfate-conjugated cellulose (DEC) beads were used for the additional depletion of LDL/VLDL. The protein expression of Apo A-I (HDL marker), Apo B (LDL/VLDL marker), and CD9 (EV marker) in supernatant and pellet was measured by ELISA. Negative staining was performed for the observation of particle shape by using transmission electron microscopy. 1) phosphotungstic acid and MgCl₂ or 2) heparin and MnCl₂ could not remove lipoproteins from plasma EV. In contrast, 3) PEG could remove HDL and most of LDL/VLDL from plasma EV. Unfortunately, 4) The DEC beads removed not only LDL/VLDL but EV from the pellets of plasma mixed with PEG. Exploring the higher yielding method for isolation of plasma EV with higher purity is required, and further research is necessary to compare this method with conventional EV isolation ones.