Aims: The present study determined the chemical compounds that may correlate with their pharmacological activities in the Flowers of Gentiana macrophylla Pall..Methods: An improved analytical method based on high performance liquid chromatography (HPLC) - photodiode array detector (DAD) was utilized to simultaneously detect and quantify iridoid glycosides (peaks IG1-IG5) and flavonoids (peaks F1-F3) in the flowers of Gentiana macrophylla Pall.,which has long been used as a traditional Tibetan medicine.Results: The calibration curves of the eight compounds showed good linearity (r2>0.9992).This method was validated to be sensitive,precise and accurate with limits of detection and quantification in range of 0.49-4.40 ng,the intra- and inter-day variations less than 1.80% for all analytes,and the overall recovery in the range of 98.93-102.42%.The application of Liquid chromatography coupled with single quadrupole mass spectrometry(MS)allowed a rapid identification of all the analytes in the mixed standard and in the crude extract of the dried flowers of Gentiana macrophylla Pall..Eight compounds (loganic acid,6’-O-β-D-glucopyranosylgentiopicroside,swertamarin,gentiopicroside,sweroside,isoorientin-4’-O-glucoside,vitexin,isoorientin) were obtained and identified as major components of the extracts.Conclusions: The analytical method with HPLC-DAD for quantitative determination was proved to be accurate and precise.According to our knowledge,this was the first report on the quantification investigation about chemical constituents in flowers of Gentiana macrophylla Pall..This study extended our understanding of this important Tibetan herbal medicine and the analytical method provided a foundation for further component analysis about Tibetan medicine.