Publisher Summary This chapter discusses the different aspects of assay for phospholipase C. Phospholipase C has become a powerful tool for studying lipid–protein interactions and membrane structure and function. Use of phospholipase C for such studies necessitates the availability of a convenient assay for enzyme activity. The substrate in radioactive assay is radioactively labeled in the phosphobase moiety. After termination of the reaction, the residual substrate is separated from the product by solvent partitioning. Enzyme activity is then measured by following either the disappearance of labeled substrate or more sensitively, the appearance of labeled product. The bioluminescence assay is based on the ability of a bacterial strain to luminesce in response to free myristic acid. Phospholipase C degrades dimyristoylphosphatidylcholine to phosphocholine and dimyristoylglycerol, which in the presence of excess pancreatic lipase, is further degraded to free myristic acid. The fluorescence of 6-carboxyfluorescein is quenched in concentrated solutions. A more conventional assay for phospholipase C must be used to establish a quantitative relationship between fluorescence measurements and levels of phospholipid hydrolyzed.