This study introduced a liquid-liquid extraction method designed for complete recovery of phospholipids from protein-rich samples, such as hemoglobin (Hb) solutions. In order to minimize protein denaturation and maximize lipid extraction from protein-rich samples, isopropanol was used as the major organic extraction solvent. In a wide range of the volume ratio of isopropanol to Hb solution at low temperatures, such extraction system resulted in limited protein precipitation and did not cause heme pigment contamination. The efficiency of phospholipid extraction from Hb solutions was pH-dependent, with the lowest extraction yield around neural pH and the highest extraction yield around pH 5.0. At pH 5.0, salt with concentrations up to 400 mmol/L in the samples did not increase extraction efficiency. Compared to other available methods, this method is simpler, needs significantly less organic solvent and, most importantly, consumed much less Hb, which is very expensive to make for the purpose of production of therapeutic products, hemoglobin-based oxygen carriers (HBOCs). This method is suitable not only for Hb solutions but also for other protein-rich biological samples, such as erythrocytes, plasma and liver.