In the present paper a study of C19-diterpene type of aconitum alkaloids, extracted from aconite roots in Aconitum carmichaeli Debx has been made using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS). The results demonstrated that the aconitum alkaloids from aconite roots can be determined simultaneously by this method, which was found to be superior to other analytical methods with regard to speed and sensitivity. Fourteen known aconitum alkaloids, including aconitines, benzoylaconitines and lipoaconitines, were assigned in the methanol extract and three compounds not reported before have been targeted separation. The evaluation of the efficiency of different extractions has been studied. These results suggested that the differences of the polarity and basicity of aconitine, and benzoylaconitines and lipoaconitines result from the C-8 constituent groups that are easily lost under MALDI.