A sensitive and rapid high-performance liquid chromatographic–tandem mass spectrometric (HPLC-MS/MS) method was developed for the quantitation of the major metabolite of aconitine, 16-O-demethylaconitine, lappaconitine as the internal standard in rat urine. Urine samples were precipitated with acetonitrile/methanol (3:1, v/v). Chromatographic separation was achieved on a Kromasil C18 analytical column. Detection was performed by a selective reaction monitoring (SRM) mode via an electrospray ionization (ESI) source operating in the positive ionization mode. The analytical method was validated in terms of specificity, precision, accuracy, extraction recovery. The intra- and inter-day precisions were less than 11.7%, and the accuracy was less than 14.0% for the analyte. The validated method has been applied to a pharmacokinetic study of 16-O-demethylaconitine in rats, following oral administration of aconitine.