Modulating the activity of lipases involved in the metabolism of plasma lipoproteins is an attractive approach for developing lipid raising/lowering therapies to treat cardiovascular disease. Identifying small molecule inhibitors for these membrane-active enzymes, however, is complicated by difficulties associated with measuring lipase activity and inhibition at the water-membrane interface; substrate and compound dynamics at the particle interface have the potential to confound data interpretation. Here, we describe a novel ELISA-based lipase activity assay that employs as "bait" a biotinylated active-site probe that irreversibly binds to the catalytic active-site serine of members of the triacylglycerol lipase family (hepatic lipase, lipoprotein lipase, and endothelial lipase) in solution with high affinity. Detection of "captured" (probe-enzyme) complexes on streptavidin-coated plates using labeled secondary antibodies to specific primary antibodies offers several advantages over conventional assays, including the ability to eliminate enzyme-particle and compound-particle effects; specifically measure lipase activity in complex mixtures in vitro; preferentially identify active-site-directed inhibitors; and distinguish between reversible and irreversible inhibitors through a simple assay modification. Using EL as an exemplar, we demonstrate the versatility of this assay both for high-throughput screening and for compound mechanism-of-action studies.