A time-resolved microPIV method is presented to measure in an EOF the particles zeta potential in situ during the transient start-up of a microdevice. The method resolves the electrophoretic velocity of fluoro-spheres used as tracer particles in microPIV. This approach exploits the short transient regime of the EOF generated after a potential drop is imposed across a microchannel and before reaching quasisteady state. During the starting of the transient regime, the electrophoretic effect is dominant in the center of the channel and the EOF is negligible. By measuring the velocity of the tracer particles with a microPIV system during that starting period, their electrophoretic velocity is obtained. The technique also resolves the temporal evolution of the EOF with three regions identified. The first region occurs before the electroosmotic effect reaches the center of the channel, the second region extends until the EOF reaches steady state, and thereafter is the third region. The two time constants separating these regions are also obtained and compared to the theory. The zeta potential of 860 nm diameter polystyrene particles is calculated for different solutions including borate buffer, sodium chloride, and deionized water. Results show that the magnitudes of the electrophoretic and electroosmotic velocities are in the range of |300| to |700| mu m/s for these measurements. The zeta potential values are compared to the well-established closed cell technique showing improved accuracy. The method also resolves the characteristic response time of the EOF, showing small but important deviations from current analytical predictions. Additionally, the measurements can be performed in situ in microfluidic devices under actual working EOF conditions and without the need for calibrations.