We have applied particle tracking techniques to obtain spatially resolved velocity measurements in electrokinetic flow devices. Both micrometer-resolution particle image velocimetry (micro-PMV) and particle tracking velocimetry (PTV) techniques have been used to quantify and study flow phenomena in electrokinetic systems applicable to microfluidic bioanalytical devices. To make the flow measurements quantitative, we performed a series of seed particle calibration experiments. First, we measure the electroosmotic wall mobility of a borosilicate rectangular capillary (40 by 400 microm) using current monitoring. In addition to this wall mobility characterization, we apply PTV to determine the electrophoretic mobilities of more than 1,000 fluorescent microsphere particles in aqueous buffer solutions. Particles from this calibrated particle/ buffer mixture are then introduced into two electrokinetic flow systems for particle tracking flow experiments. In these experiments, we use micro-PIV, together with an electric field prediction, to obtain electroosmotic flow bulk fluid velocity measurements. The first example flow system is a microchannel intersection where we demonstrate a detailed documentation of the similitude between the electrical fields and the velocity fields in an electrokinetic system with uniform zeta potential, zeta. In the second system, we apply micro-PIV to a microchannel system with nonuniform zeta. The latter experiment provides a simultaneous measurement of two distinct wall mobilities within the microchannel.