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Electrokinetic properties such as the mobility, surface charge, and zeta potential of sub-millimeter particles are vital parameters in various industrial applications. Their measurement and control in aqueous media have been extensively studied. However, despite their growing importance, the electrokinetic properties of organic solvents have not been studied as thoroughly as those of aqueous media. An electrophoresis cell with a microscope monitor was designed for the electrokinetic studies of sub-millimeter particles in cyclohexane, which is a solvent with very low permittivity. The movement of large particles in the range of 4 ~ 478 µm was successfully traced under a strong electric voltage up to 1100 V, even without the addition of surfactants. The particle sizes were at least 300 times larger than those reported previously. By applying electric fields up to 55 kV/m, the electrophoretic mobilities were measured to be of the order of 10 -9 to 10 -7 m 2 /V∙s through image processing of the recorded particle movement. Five organic sub-millimeter particles had charge densities in the range of -3.5 ~ 4.4 e/µm 2 , and polyethersulfone particles showed extremely high mobilities. The surface charge of organic and inorganic particles is mainly generated by the dissociation of hydroxide groups or by the protonation to surface Lewis base oxygen atoms.