Safflomide (N-caffeoyltryptamine) is a compound belonging to a group of phenylpropanoid amides found in plants. In this study, safflomide was chemically synthesized and confirmed by LC–MS, LC–MS/MS and NMR spectroscopic methods, and a high-performance liquid chromatography (HPLC) method was developed for quantifying safflomide in biological samples. The synthesis was simple, and the yield of safflomide was greater than 50%. Using the synthesized safflomide as a standard, HPLC separation was performed on a Nova-Pak C18 column using an isocratic buffer, and the separation was detected using a coulometric electrochemical detector. The detection of safflomide yielded an excellent peak resolution at the retention time of 21min, and the lower limit of the detection was as little as 100fmol. Using this HPLC method, the plasma concentrations of safflomide were determined in mouse blood, collected at 5, 10, 15, 20, 25, 30, and 35min following its oral administrations (1 and 3mg/30g body weight). This HPLC method standardized with safflomide is the first reported method able to quantify the compound in standard and plasma samples with good detection limit and consistent reproducibility.