This work presents theoretical, numerical and experimental investigations of electrokinetic transport and separation of droplets in a microchannel. A theoretical model is used to predict that, in case of micron-sized droplets transported by electro-osmotic flow, the drag force is dominant as compared to the dielectrophoretic force. Numerical simulations were performed to capture the transient electrokinetic motion of the droplets using a two-dimensional multi-physics model. The numerical model employs Navier-Stokes equations for the fluid flow and Laplace equation for the electric potential in an Arbitrary Lagrangian-Eulerian framework. A microfluidic chip was fabricated using micromilling followed by solvent-assisted bonding. Experiments were performed with oil-in-water droplets produced using a cross-junction structure and applying electric fields using two cylindrical electrodes located at both ends of a straight microchannel. Droplets of different sizes were produced by controlling the relative flow rates of the discrete and continuous phases and separated along the channel due to the competition between the hydrodynamic and electrical forces. The numerical predictions of the particle transport are in quantitative agreement with the experimental results. The work reported here can be useful for separation and probing of individual biological cells for lab-on-chip applications. © 2013 Springer-Verlag Berlin Heidelberg.