Abstract: A method of TLC densitometry was established in order to determine the main alkaloids: mesaconitine, aconitine and hypaconitine in Aconite. root. The powdered sample was alkalinized by ammonia and macerated with ether for 24 hThe mixture was then centrifuged, the residue was washed three times each with 2 ml of fresh ether, the combined ether extract was evaporated to dryness and then dissolved in 1 ml of dichloromethane.Standard solution and sample solution were spotted on a silica gel GF254 plate, and developed with cyclohexane-ethyl acetate-diethylamine (8:1: 1),the chromatogram was observed under UV light as dark spots. A Shimadzu TLC model 910 was used for scanning at λs 236 nm and λR 350nm by reflection mode. Linear calibration curves were obtained for the 3 constituents in the range of 2～6 μg. The average recoveries of masaconitine, aconitine and hypaconitine were 97.3, 96.4, 99.1% and the variation coefficients were 1.81, 1.72, 1.18%, respectively. The spots were stable for more than 24 h.Samples from various sources were analyzed.