OBJECTIVES The standard methods for quantifying fat absorption involve extraction of fat from fecal samples with heptane, ether and ethanol. These solvents do not quantitatively recover phospholipids. Malabsorption of dietary and biliary phosphatidylcholine could potentially result in choline deficiency. Therefore, the authors developed a method extracting and quantifying fecal phospholipids. METHODS Fecal samples were collected for 72 hours from 18 children with cystic fibrosis and 10 control children. Fat was extracted first with hexane/diethyl ether/ethanol and then with chloroform/methanol. Total fat was quantitated gravimetrically. Phospholipids in extracted fat were separated and quantified using high-performance liquid chromatography with evaporative light-scattering detection (HPLC-ELSD). Phospholipid quantification was validated with a phosphomolybdate colorimetric assay. RESULTS The combination of solvent systems used in this study significantly improved total fat (p < 0.05) and phospholipid (p < 0.001) extraction compared with either hexane/diethyl ether/ethanol or chloroform/methanol alone. Fecal phospholipid measured by HPLC-ELSD was significantly correlated with lipid-soluble phosphorous using the phosphomolybdate assay (r = 0.75, p < 0.001). This method also allows quantification of fecal phosphatidylcholine and lysophosphatidylcholine. CONCLUSIONS Hexane/diethyl ether/ethanol followed by chloroform/methanol extraction of fecal samples and quantification of phospholipids using HPLC-ELSD is a new method for investigating phospholipid malabsorption.