An analytical method incorporating high-performance liquid chromatography (HPLC) with MS and UV-detection was developed for the qualitative and quantitative determination of alkaloids in Corydalis yanhusuo. Ten alkaloids, including seven tertiary alkaloids and three quaternary alkaloids, were identified by comparing their retention times, UV and MS spectra with those of authentic compounds. Furthermore, the collision-induced dissociations of the [M+H](+) and [M](+) ions were studied to clarify the MS behavior of the different types of alkaloids. In positive ion electrospray ionization tandem mass spectrometry (ESI-MS) all the tertiary alkaloids yielded prominent [M+H](+) ions and quaternary alkaloids yielded prominent [M](+) ions in the first order mass spectra. Fragments involving losses of H, CH(3), CO, H(2)O and OCH(3) were observed in the MS/MS spectra. In addition, quantification of the 10 alkaloids in Corydalis yanhusuo from methanol and ethyl acetate extract of different origins were performed by this method, which provides a new tool for the assessment of quality of Corydalis yanhusuo preparations. The method provides the best sensitivity and specificity for characterization and quantitative determination of the alkaloids in Corydalis yanhusuo so far.