Abstract The preparation of phosphatidyl- p -nitrophenol and its use as a substrate in the non-enzyme based assay for the quantitative evaluation of phospholipase D activity is described. The solution of the substrate in Tris buffer pH 8 and Triton X-100 is mixed directly with an aliquot of a fermentation broth or a plant extract and the absorbance of the solution at 405 nm is read. The method is rapid, accurate and inexpensive. Application in the pH range 4–9 is possible.