The objective of this study was to develop a new protocol that could be used for large-scale separation of phosvitin from egg yolk using ethanol and salts. Yolk granules, which contain phosvitin, were precipitated after diluting egg yolk with 9 volumes of distilled water. The pH of the yolk solution was adjusted to pH 4.0 to 8.0 using 6 N HCl or NaOH, and then yolk granules containing phosvitin was separated by centrifugation at 3,220 × g for 30 min. Lipids and phospholipids were removed from the insoluble yolk granules using 85% ethanol. The optimal volumes and concentration of ethanol in removing lipids from the precipitants were determined. After centrifugation, the lipid-free precipitants were homogenized with 9 volumes of ammonium sulfate [(NH(4))(2)SO(4)] or NaCl to extract phosvitin. The optimal pH and concentration of (NH(4))(2)SO(4) or NaCl for the highest recovery rate and purity for phosvitin in final solution were determined. At pH 6.0, all the phosvitin in diluted egg yolk solution was precipitated. Among the (NH(4))(2)SO(4) and NaCl conditions tested, 10% (NH(4))(2)SO(4) or 10% NaCl at pH 4.0 yielded the greatest phosvitin extraction from the lipid-free precipitants. The recovery rates of phosvitin using (NH(4))(2)SO(4) and NaCl were 72 and 97%, respectively, and their purity was approximately 85%. Salt was removed from the extract using ultrafiltration. The salt-free phosvitin solution was concentrated using ultrafiltration, the impurities were removed by centrifugation, and the resulting solution was freeze-dried. The partially purified phosvitin was suitable for human use because ethanol was the only solvent used to remove lipids, (NH(4))(2)SO(4) or NaCl was used to extract phosvitin, and ultrafiltration was used to remove salt and concentrate the extract. The developed method was simple and suitable for a large-scale preparation of partially purified phosvitin.