Triboelectric charging is a potentially suitable tool for separating fine dry powders, but the charging process is not yet completely understood. Although physical descriptions of triboelectric charging have been proposed, these proposals generally assume the standard conditions of particles and surfaces without considering dispersity. To better understand the influence of particle charge on particle size distribution, we determined the in situ particle size in a protein−starch mixture injected into a separation chamber. The particle size distribution of the mixture was determined near the electrodes at different distances from the separation chamber inlet. The particle size decreased along both electrodes, indicating a higher protein than starch content near the electrodes. Moreover, the height distribution of the powder deposition and protein content along the electrodes were determined in further experiments, and the minimum charge of a particle that ensures its separation in a given region of the separation chamber was determined in a computational fluid dynamics simulation. According to the results, the charge on the particles is distributed and apparently independent of particle size.