Particle characterization techniques are essential in a wide range of fields, including biological sciences, material science, environmental studies, pharmaceutical, and clinical research. Despite the availability of numerous characterization methods, spanning both ensemble and single particle characterization techniques, there remains a critical need for more precise and robust methods capable of multiparameter characterization at the single-particle level, particularly for heterogeneous biological particles. This dissertation aims to address this gap by developing a novel microfluidic system that combines low-frequency (1-100 Hz) AC electrophoresis with particle tracking velocimetry (PTV), enabling comprehensive multiparameter characterization of single particles and cells. All TrACE devices utilize low-frequency AC electrophoresis within microfluidic channels to manipulate particles and cells based on their electrophoretic mobility. When combined with PTV, this system tracks the motion of individual particles in solution over time, allowing for the determination of their physical properties based on their unique trajectories. Because low-frequency AC electrophoresis remains underutilized, the underlying mechanisms of electromigration in low frequency electric field waves have received limited attention. To address this, a combined experimental and theoretical approach is employed to measure and model both particle and cell trajectories. 1D finite element modeling has provided fundamental insights into the mechanisms driving particle migration, with predicted particle trajectories showing good agreement with measured particle trajectories. The performance of the TrACE system is thoroughly characterized using commercially available polystyrene particles, demonstrating its ability to simultaneously measure particle size and electrophoretic mobility. The second version of TrACE, known as high-voltage (HV)-TrACE, expands the system’s capabilities by incorporating a new device design that applies voltages above the water electrolysis threshold. The application of higher voltages increases electrophoresis, leading to improved measurement precision and extended particle excursions during each half-period compared to the original TrACE. The electrophoretic particle transport in HV-TrACE can also be utilized in particle-particle binding assays. By significantly overcoming the transport limitations imposed by Brownian motion, HV-TrACE enables more effective mixing of particles with different electrophoretic mobilities and allows for real-time monitoring of particle-particle binding in solution. In addition, HV-TrACE can measure the physical properties of single cells and track them throughout the binding process, enabling the characterization of cellular subpopulations with distinct receptor distributions. We envision that this newly developed multiparameter characterization system will have a significant impact on the fields of single-particle analysis and single-cell phenotyping. By providing insights into fundamental properties such as particle size, charge, and shape, it has the potential to advance research across a wide range of scientific disciplines.