This paper introduces the concept of a ratcheting electrophoresis microchip (REM), a microfluidic device for electrophoretic separation of macromolecules such as DNA and proteins in aqueous solution using low applied voltages (∼ 1 V). The device consists of several thousands of parallel linear electrodes with a constant pitch of about 10 μm. A spatial sawtooth like potential distribution generated by the electrode array causes local electrophoretic migration of charged molecules between adjacent electrodes. By cycling the potential distribution in a certain pattern, the spatio-temporal electrophoretic ratchet can be used to separate and manipulate macromolecules at speeds much faster than thermal ratchets or more traditional techniques such as capillary or gel electrophoresis. This paper describes results of two simulations: First, using a simple one-dimensional potential distribution for the ratchet, the basic device function is examined using a probabilistic approach that simulates the interplay between electrophoretic mobility and molecular diffusion. The results suggest that the REM can function as a molecular filter through which only molecules having mobility larger than a threshold can pass. The REM can also be programmed to separate molecules to create a molecular profile, much like conventional electrophoresis. Second, two-dimensional stochastic simulations based on molecular diffusion and transient Debye screening by mobile ions are used to demonstrate the feasibility of the REM. The results suggest that biomolecular separation can indeed be achieved within time and length scales much shorter than capillary and gel electrophoresis.