Insulator-based dielectrophoresis (iDEP) employs insulating structures embedded in a microchannel to produce electric field gradients. This contribution presents a detailed analysis of the regions within an iDEP system where particles are likely to be retained due to dielectrophoretic trapping in a microchannel with an array of cylindrical insulating structures. The effects of particle size and shape on dielectrophoretic trapping were analyzed by employing 1 and 2 m polystyrene particles and Escherichia coli cells. This research aims to study the mechanism behind dielectrophoretic trapping and develop a deeper understanding of iDEP systems. Mathematical modeling with COMSOL Multiphysics was employed to assess electrokinetic and dielectrophoretic particle velocities. Experiments were carried out to determine the location of dielectrophoretic barriers that block particle motion within an iDEP microchannel; this supported the estimation of a correction factor to match experiments and simulations. Particle velocities were predicted with the model, demonstrating how the different forces acting on the particles are in equilibrium when particle trapping occurs. The results showed that particle size and shape have a significant effect on the magnitude, location, and shape of the regions of dielectrophoretic trapping of particles, which are defined by DEP isovelocity lines and EK isovelocity lines.