The objective of this study was to develop a simple, sequential separation method for multiple proteins from egg white. Separated proteins are targeted for human use, and thus any toxic compounds were excluded. The methods for individual components and the sequential separation were practiced in laboratory scale first, and then tested for scale-up. Lysozyme was separated first using a cation exchange resin and then ovomucin using isoelectric precipitation. Ovalbumin and ovotransferrin were separated from the lysozyme- and ovomucin-free egg white by precipitating ovotransferrin twice using (NH4)2SO4 and citric acid combination. After centrifugation, the supernatants were used for ovalbumin separation. The precipitants were used as ovotransferrin fraction, and the supernatant was desalted using ultrafiltration, and then heat-treated to remove impurities. The yield of ovomucin and ovalbumen was > 98% and that of ovotransferrin and lysozyme was > 82% for both laboratory and scale-up preparations. SDS-PAGE and Western Blotting of the separated proteins, except for ovomucin, showed > 90% purity and the activities of separated ovalbumin, ovotransferrin, and lysozyme were > 96%. The protocol separated four major proteins in sequence, and the method was simple and easily scaled-up. The separated proteins can be used as functional components.