Impurities in chemically synthesized auraptene, an active pharmaceutical ingredient (API), were detected by a gradient Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) method. Molecular weights and major product ions of these chemical compounds were determined by Liquid Chromatography/Triple Quadrupole (LC–MS/MS) analysis. Structural assignments were presumed as umbelliferone (Imp-I), (E)-6,7-dihydroxy-3,7-dimethyl-2-octene-umbelliferone (Imp-II), (E)-6,7-epoxy-3,7-dimethyl-2-octene-umbelliferone (Imp-III) and 4-methylauraptene (Imp-IV). The impurities were authentically synthesized, confirmed by Nuclear Magnetic Resonance spectroscopy (NMR) and Infrared spectroscopy (IR), and subsequently used as reference samples in routing HPLC system suitability testing for method specificity and detectability. Method specificity was further verified by forced degradation studies. The developed method was validated for characterization of impurities in synthesized auraptene according to the guidelines of the International Conference on Harmonization (ICH) in our laboratory.