A novel approach to molecular separations is investigated using a technique termed droplet-based isoelectric focusing. Drops are manipulated discretely on a superhydrophobic surface, subjected to low voltages for isoelectric focusing, and split-resulting in a preparative separation. A universal indicator dye demonstrates the generation of stable, reversible pH gradients (3-10) in ampholyte buffers, and these gradients lead to protein focusing within the drop length. Focusing was visually characterized, spectroscopically verified, and assessed quantitatively by noninvasive light scattering measurements. It was found to correlate with a quantitative model based on 1D steady-state theory. This work illustrates that molecular separations can be deployed within a single open drop, and the differential fractions can be separated into new discrete liquid elements.