Field plants such as legumes and cereal are known as primary sources of essential nutrients such as protein. Food products enriched with plant proteins have been considered a vital resource to meet the nutritional requirements of the increasing global population. Plant proteins consist of polypeptide chains comprising different three-dimensional structures of protein fractions. These supramolecular structures determine the highly specific activities and techno-functionality possessed by proteins. Characterization of the protein fraction profile is essential for evaluating structural, functional, and nutritional values of plant proteins. These characteristics are also important for food formulations in the food industry. Plant proteins are classified based on their solubility in aqueous-based solutions. This system divides proteins into generalized categories called Osborne fractions. Albumin is soluble in water, globulin in dilute saline solutions, prolamin in aqueous ethanol solutions, and glutelin in acidic or alkaline solutions. This chapter outlines a clear procedure for Osborne fractionation as a promising approach to the characterization of plant proteins.