Phospholipase D (PLD) hydrolyzes structural phospholipids like phosphatidylcholine (PC) and phosphatidylethanolamine (PE) into phosphatidic acid (PA) and free choline/ethanolamine. In plants, this activity can be stimulated by a wide variety of biotic and abiotic stresses (Li et al., Biochim Biophys Acta 1791:927–935, 2009; Testerink and Munnik, J Exp Bot 62(7):2349–2361, 2011). This chapter describes a protocol for the measurement of PLD activity in vivo. The protocol takes advantage of a unique property of PLD, i.e., its ability to substitute a primary alcohol, such as 1-butanol, for water in the hydrolytic reaction. This transphosphatidylation reaction results in the formation of phosphatidylbutanol (PBut), which is a specific and unique reporter for PLD activity. The assay is highly sensitive for detecting PLD activity in vivo, following stimulation of intact plant cells, seedlings, and tissues, being a valuable method for studying the regulation of plant PLD activity in vivo.