This paper is to report the analysis of the main chemical constituents of Shuanghuanglian injection powder and determination of their origin. The sample solution was analyzed by a Zorbax C18 column with a gradient mobile phase comprised of methanol and 0.25% acetic acid solution. Both UV and electrospray ionization mass spectrometry detector were used simultaneously, -Q1-scan detection mode was evaluated for the identification of the LC peaks. To analyze the mass spectrum of every LC peaks, 43 molecular mass from the ion chromatogram of Shuanghuanglian injection powder were identified and among them, structure of 20 compounds were elucidated, and the data were sorted to the three component herbs, separately.