Over the past decade, tremendous progress has been made toward understanding the physiological functions of individual members of the diverse phospholipase D (PLD) family of enzymes in plants. For instance, the involvement of plant PLD members has been shown or suggested in a wide variety of the cellular and physiological processes such as regulating stomatal opening and closure; signaling plant responses to drought, salt, and other abiotic and biotic stresses; organizing microtubule and actin cytoskeletal structures; promoting pollen tube growth; cycling phosphorus; signaling nitrogen availability; regulating N-acylethanolamine stress signaling; and remodeling membrane phospholipids in plant responses to phosphate deprivation and during and after freezing. There are at least a dozen PLDs in Arabidopsis that can be separated into six classes, phospholipases Dα, Dβ, Dγ, Dδ, Dε, and Dζ, based on their molecular and enzymatic characteristics. Several of the classes have distinguishing enzymatic properties that can be used to discriminate among the various classes. Here we provide four variations of in vitro PLD activity assays using choline-labeled phosphatidylcholine to distinguish, to the extent possible, among the different PLD classes.