New and important separations capabilities are being enabled by utilizing other electric field-induced forces besides electrophoresis, among these is dielectrophoresis. Recent works have used experimentally simple insulator-based systems that induce field gradients creating dielectrophoretic force in useful formats. Among these, juxtaposing forces can generate gradient-based steady-state separations schemes globally similar to isoelectric focusing. The system of interest is termed gradient insulator-based dielectrophoresis and can create extremely high resolution steady-state separations for particles four nanometers to ten micrometers in diameter, including nearly all important bioparticles (large proteins, protein aggregates, polynucleotides viruses, organelles, cells, bacteria, etc.). A theoretical underpinning is developed here to understand the relationship between experimental parameters and resolution and to identify the best expected resolution possible. According to the results, differences in particles (and bioparticles) as small as one part in 10[superscript 4] for diameter (subnanometer resolution for a one micrometer particle), one part in 10[superscript 8] for dielectrophoretic parameters (dielectrophoretic mobility, Clausius–Mossotti factor), and one part in 10[superscript 5] for electrophoretic mobility can be resolved. These figures of merit are generally better than any competing technique, in some cases by orders of magnitude. This performance is enabled by very strong focusing forces associated with localized gradients.