Increasing evidence of phospholipid peroxidation in the pathophysiology of various diseases demonstrates the need for pure phosphatidylcholine hydroperoxide (PCOOH) reference as a standard for quantification, and for biological studies in this field. Our previous study showed the usability of reaction between hydroperoxide and vinyl ether for preparation of PCOOH. However, the reaction has sometimes provided brown byproducts. Here, we report a method to improve reaction conditions, and demonstrate the production of 30 mg of PCOOH (a pure mixture of 13- and 9-hydroperoxyoctadecadienoyl PCOOH isomers) from 100 mg of phosphatidylcholine without byproduct formation. The resultant PCOOH will be useful as a standard for quantification studies, as well as for the evaluation of PCOOH pathogenicity.