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The ability to measure the charge and size of single particles is essential to understanding particle adhesion and interaction with their environment. Characterizing the physical properties of biological particles, like cells, can be a powerful tool in studying the association between the changes in physical properties and disease development. Currently, measuring charge via the electrophoretic mobility (μep) of individual particles remains challenging, and there is only one prior report of simultaneously measuring μep and size. We introduce microfluidic transverse AC electrophoresis (TrACE), a novel technique that combines particle tracking velocimetry (PTV) and AC electrophoresis. In TrACE, electric waves with 0.75 to 1.5 V amplitude are applied transversely to the bulk flow and cause the particles to oscillate. PTV records the particles' oscillating trajectories as pressure drives bulk flow through the microchannel. A simple quasi-equilibrium model agrees well with experimental measurements of frequency, amplitude, and phase, indicating that particle motion is largely described by DC electrophoresis. The measured μep of polystyrene particles (0.53, 0.84, 1, and 2 μm diameter) are consistent with ELS measurements, and precision is enhanced by averaging ∼100 measurements per particle. Particle size is simultaneously measured from Brownian motion quantified from the trajectory for particles <2 μm or image analysis for particles ≥2 μm. Lastly, the ability to analyze intact mammalian cells is demonstrated with B cells. TrACE systems are expected to be highly suitable as fieldable tools to measure the μep and size of a broad range of individual particles.