Abstract: OBJECTIVE To develop an HPLC-ESI/MS method for the identification of the fingerprint characteristic peak of Jiweiling freeze-dried powder. METHODS With a Symmetry C18 column(4.6 mm×250 mm, 5 μm), acetonitrile-water-1% acetic acid was used as mobile phase at a flow rate of 1.0 mL·min^-1, eluted in gradient mode and column temperature maintained at 30 ℃. The sample was determined by mass spectrum with API-ESI ionization source. The pressure of atomization room was 275.8 kPa, the pressure of adjunctive gas was 137.9 kPa, the temperature of capillary was 340 ℃, the atomization voltage was 4.2 kV. The positive and negative canner mode was selected, the total ion current(TIC) chromatography and characteristic fragment ions were recorded. RESULTS Twelve major chromatographic peaks were marked in the fingerprint characteristic peak of Jiweiling freeze-dried powder. Seven chromatographic peaks were founded in Ginseng Radix et Rhizoma, five chromatographic peaks were founded in Epimedii Folium. CONCLUSION The chemical constituents and the sources of Jiweiling freeze-dried powder is identified. And it is quite valuable for the quality evaluation as well as chemical characterization of Jiweiling freeze-dried powder.