Attempts to characterize, quantify, and/or modulate the activity of the secreted phospholipase A, family of enzymes result from the diversity of physiological roles for which these enzymes have been implicated. The 1-palmitoyl-2-(10-pyrenedecanoyl)-phosphatidylglycerol (pyrenePG)-based fluorometric assay is a sensitive and readily adaptable method for further elucidating phospholipase function under various experimental conditions, as well as a tool for screening chemical libraries for potent inhibitors of this enzymatic activity. This assay is based on the observed difference in fluorescent emission of pyrene aggregated in vesicles compared to sequestered in monomeric form by binding to bovine serum albumin after lipolytic activity, thus allowing direct quantification of hydrolyzed fatty acids by the measurement of the corresponding monomeric emission intensity. The assay can be carried out in multiwell plates for high-throughput screening of compound libraries.