Kangshen granule, containing 12 kinds of traditional Chinese medicine, has complex composition. A new method for the determination of puerarin in Kangshen granule was developed using reversed-phase high performance liquid chromatography coupled with matrix assisted laser desorption ionization time of flight mass spectrometry and ultraviolet detection (RP-HPLC/MALDI-TOF MS/UV). The sample was separated by RP-HPLC with a non-linear gradient elution, and then was qualitatively detected by mass spectrometry to identify the puerarin peak. The chromatographic process was performed on a YMC-ODS column under the optimized conditions with a mobile phase consisting of methanol, water and 0.1% TFA, and detected at 250 nm. The linear range of puerarin was from 0. 132 to 1.32 microg (r = 0.9997). The recovery was higher than 103% and the relative standard deviation was lower than 2.0% (n=6). The puerarin content in Kangshen granule sample was determined and the puerarin content was 3.09 mg/g. The method is simple and feasible for the determination of puerarin with a good repeatability. The method can be used for quality control of Kangshen granule.