Air classification of an industrial barley endosperm fraction with initial protein content of 8.3% mixed with Aerosil 200F flow aid resulted in protein enrichment up to 28.3% with 21.7% protein separation efficiency. By reducing the classifier wheel speed from 21500 rpm to 8000 rpm, the protein separation efficiency increased to 59.4%, whereas the protein content only decreased to 22.3%. Microstructural analysis showed that small starch granules separated together with protein in the fine fraction and had more continuous protein matrix around them. The protein-enriched fraction obtained at 8000 rpm classifier wheel speed had limited protein solubility of 9.3% at native pH 5.1. Solubility increased in acidic (pH 3, 19.6%) and especially in alkaline (pH 11, 91.9%) conditions. Protein fraction had water and oil binding capacities of 1.2 and 1.1 g/g, respectively. Foaming properties tested at different pH-values revealed best capacity at pH 3. Thermograms showed two melting temperatures, varying from 60.0 to 64.2 °C and from 90.5 to 95.5 °C for all the studied fractions. Cereal side-streams such as the industrial barley endosperm fraction can be utilised as potential plant protein ingredient for various food/feed applications. However, further studies are needed to evaluate the technological, nutritional, and sensory characteristics of this protein enriched fraction.