We report a microfabricated field flow fractionation device for continuous separation of subcellular organelles by isoelectric focusing. The microdevice provides fast separation in very small samples while avoiding large voltages and heating effects typically associated with conventional electrophoresis-based devices. The basis of the separation is the presence of membrane proteins that give rise to the effective isoelectric points of the organelles. Simulations of isoelectric focusing of mitochondria in microchannels are used to assess design parameters, such as dimensions and time scales. In addition, a model of Joule heating effects in the microdevice during operation indicates that there is no significant heating, even without active cooling. The device is fabricated using a combination of photolithography, thin-film metal deposition/patterning, and electroplating techniques. We demonstrate that in the microfluidic devices, mitochondria from cultured cells migrate under the influence of an electric field into a focused band in less than 6 min, consistent with model predictions. We also illustrate separation of mitochondria from whole cells and nuclei as well as the separation of two mitochondrial subpopulations. When automated and operated in parallel, these microdevices should facilitate high-throughput analysis in studies requiring separation of organelles.