A micron-resolution particle image velocimetry (PIV) system has been developed to spatially and temporally resolve electroosmotic flow fields within microfluidic bioanalytical devices. A second diagnostic technique, particle tracking velocimetry (PTV) has been used to determine the distribution of electrophoretic mobilities of seed particles and thereby make the PIV measurements quantitative. This second particle tracking technique has been used to determine probability distribution functions of the seed particles. Results from simulations of electric fields yield local electric field strengths in the geometries of interest. The measured mean mobility of the seed particles (obtained from PTV measurements) is then multiplied by the local electric field vector to obtain the electrophoretic velocity. The variance on the particle mobility measurement influences the errors introduced in the electroosmotic flow measurements. After total particle velocities are measured within a microfluidic system of interest, the seed particle electrophoretic velocities are subtracted from the PIV total velocity data to obtain electroosmotic flow field velocities. Ensemble-averaged velocity field measurements for electroosmotic flow at the intersection of a cross-channel are presented.