An advanced and reliable high performance liquid chromatography (HPLC)/ultraviolet detector (UV)/ion-trap timeof-flight (IT-TOF) mass spectrometry was developed for the simultaneous quantification of 19 marker compounds in Bangpoong-tong-sung-san (BPTS), a traditional oriental prescription. Various parameters affecting HPLC separation and IT-TOFdetection were investigated, and optimized conditions were identified. The separation was achieved on a Capcell PAK C18 column(1.5 mm × 250 mm, 5 μm particle size) using a gradient elution of acetonitrile and water containing 0.1% formic acid at aflow rate of 0.1 mL/min. The column temperature was maintained at 40oC and the injection volume was 2 μL. IT-TOF systemwas equipped with an electrospray ion source (ESI) operating in positive or negative ion mode. The optimized electrospray ionizationparameters were as follows: ion spray voltage, +4.5 kV (positive ion mode), or -3.5 kV (negative ion mode); drying gas(N2), 1.5 L/min; heat block temperature, 200oC. Automatic MSn (n = 1~3) analyses were carried out to obtain structural informationof analytes. Elemental compositions and their mass errors were calculated based on their accurate masses obtained from aformula predictor software. The marker compounds in BPTS were identified by comparisons between MSn spectra from standardsand those from extracts. Moreover, the libraries of MS2 and MS3 spectra and accurate masses of parent and fragment ionsfor marker compounds were constructed. The developed method was successfully applied to the BPTS extracts and identified 17out of 19 marker compounds in the BPTS extracts.