Phospholipases of the A type constitute a large family of esterases that catalyze the hydrolysis of the fatty acid ester bonds in phospholipids and thus generate lysophospholipids and fatty acids. Both products or their metabolites are important signal molecules in the cellular adaptation to stress, developmental processes and several diseases in plants and animals. The assay of PLA activity has been much promoted by the availability of phospholipid substrates with fluorophores at one or two fatty acids. The double labeled compounds display an increase of fluorescence due to the escape from intramolecular quenching or FRET. They thus allow the sensitive monitoring of PLA activity even without a separation of the hydrolysis products. This chapter is focused on the proper use of fluorescent (BODIPY) labelled substrates for assays of PLA activity in cells and subcellular fractions by fluorimetric analysis and classical or confocal microscopy.