Current methods for measuring lung lipids obtained by bronchoalveolar lavage are time consuming and require sample extraction with organic solvents. Here we utilized enzymatic methods for measuring the major phospholipid classes found in human bronchoalveolar lavage fluid (BALF), namely phosphatidylcholine and phosphatidylglycerol, as well as the neutral lipid cholesterol. These assays can be carried out on as little as 200 microliters lavage fluid in 96-well microtiter plates without the need for organic solvents. Results were verified by comparison with HPLC and chemical methods. The measured values by all three methods were in agreement with previous studies in which lipid analysis was performed by thin-layer chromatography. By contrast to thin-layer chromatography, however, the methods described here can be efficiently performed with small quantities of material without sacrificing accuracy. This methodology can facilitate the characterization of the major surfactant-associated lipids in BALF and foster improved understanding of the role of these lipids in human lung disease.