[Background and Purpose] Huperzia serrata Thunb.ex Murray (Qian Ceng Ta in Chinese), a perennial herb, has been used as a traditional Chinese folk medicine for centuries for the treatment of contusion, strain, swelling and schizophrenia.In the 1980s, Huperzine A (Hup A) and Huperzine B (Hup B) were isolated from this plant.Since Huperzine A (HupA), Huperzine B (HupB) and Huperzine C (HupC) have proved to be potent, reversible and selective inhibitors for AchE activity, H.serrata has captured much attention of researchers and medical professionals from all over the world.H.serrata is distributed mainly in the areas along the Yangtze River and throughout the southern parts of China.Previous studies indicated that the content of Hup A is very low in H.serrata and higher in leaves than other parts.However, the distribution of Hup A, Hup B and Hup C in H.serrata from Hainan is not under investigation.Currently, various analytical methods for Hup A in H.serrate can be found;however, there are few methods to simultaneously determine Hup A, Hup B and Hup C.In the current study, a reliable method has been developed and validated for quantification of three alkaloids (Hup A, Hup B and Hup C) from H.serrata based on ultraperformance liquid chromatography (UPLC) with a photodiode array detector (PDA) and confirmed by tandem quadrupole time-of-flight mass spectrometry (Q/TOF-MS).