Two-Color μ-PIV is developed and used to uniquely determine the fluid velocity based on the micron-resolution Particle Image Velocimetry (μ-PIV) technique [1–3]. The fluid velocity field was obtained by measuring the motion of two different sizes particles, 0.7 and 1.0 μm. The different sizes of particles contain different fluorescent dyes, allowing them to be distinguished using fluorescent filter cubes. By comparing the velocity fields from the two different size particles, the underlying fluid motion can be uniquely determined, without a priori knowledge of the electrical properties of the particles, or the electrical field. The test section is formed by two wedge-shaped electrodes sandwiched between two glass wafers. In the presence of nonuniform ac electric fields, the particles experience dielectrophoretic (DEP) forces due to polarization and drag forces due to viscous interaction with the suspending medium, and the fluid motion is induced by the electrothermal effect and/or ac electroosmosis. The micro-PIV measurements are used to determine quantitatively the physical characteristics of the AC electrokinetic effects.