The electroosmotic flow in laminated excimer laser-ablated microchannels has been studied as a function of the depth of the rectangular channels, and particular emphasis has been given to the difference in the ζ-potentials between the lamination layer and the ablated substrate. Experimental electroosmotic flow follows the tendency predicted by a recently published model. The ζ-potentials of lamination and ablated surfaces were determined for poly(ethylene terephthalate) and poly(carbonate) substrates by fitting the experimental data with a numerical implementation of this model. In the experimentally investigated range of channel cross sections, a linear fit to the data gives a good approximation of the ζ-potentials for both materials. Moreover, a flow injection analysis of fluorescein dye has been performed to show the severe loss in numbers of theoretical plates, caused by Taylor dispersion, when such microchannels, dedicated to microcapillary electrophoresis, are used.