Supercritical carbon dioxide (SC-CO2) is very effective in removing oils from a variety of seed matrices, devoid of any appreciable amount of phospholipid (PL) content. However. the limited solubility of PLs in SC-CO2 leaves behind a potentially valuable by-product in the spent seed matrix. Any PL extraction process from the spent matrix must maintain the structure and the quality of PLs and must be compatible with the end use of the seed protein meal as animal feed or for human consumption. An initial SC-CO2 extraction of soybean flakes was performed at 32 MPa and 80 degrees C to extract the oil, leaving the PLs in the defatted soybean flakes (DSF). A second step was performed on the DSF using X-eth =0.10, varying the pressure from 16.6 to 68.4 MPa and the temperature from 60 to 80 degrees C. For all SC-CO2/ethanol extractions, a fraction rich in PLs was obtained. The fractions extracted from defatted soybean flakes were dried and then re-dissolved in chloroform before HPLC-ELSD analysis. Quantitative and qualitative analysis of PLs on soybean seeds, DSF, and different extracted phospholipid fractions was carried out, to ascertain the effort of combinations of extraction pressure and temperature.