Acetophenones in Cynanchum species, especially cynandione A and its derivatives, whose utilization and toxicity in herbal drugs and folk medicines has caused great interest in the chemical investigation, have extensive biological activities. In this paper, a facile method based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS(n)) was developed for the analysis of cynandione A derivatives in the roots of the Cynanchum wilfordii and C. auriculatum. ESI-MS/MS and ESI-MS(n) analysis of cynandiones A and B in negative ion mode were firstly performed employing two mass spectrometers each equipped with an ion-trap and a quadrupole time-of-flight (Q-TOF) mass analyzer. The results drawn from both instruments were similar to each other. Characteristic fragmentation pathways were proposed by comparing the spectra of two standards acquired in the experiments. The fragment ions at m/z 283 and 268 were obtained, and then were used as diagnostic ions to screen and identify cynandione A derivatives from the roots of above two species, together with an HPLC-MS(n) method. Total of 28 cynandione A derivatives comprising 4 reported and 24 novel components were identified or tentatively identified. Furthermore, breakdown curves were constructed to distinguish two types of isomers among these compounds. To our knowledge, this is the first report on characterization of acetophenones by HPLC-ESI-MS(n), which allows a rapid and complete analysis of cynandione A derivatives in roots of Cynanchum species.