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Introduction: Selaginella species have been used widely for the treatment of inflammatory diseases, diabetes and cancer. In this study, the contents of the total flavonoids and amentoflavone in five Selaginella plant drugs [Selaginella moellendorffii Hieron. (SM), Selaginella delicatula (Desv.) Alston (SD), Selaginella uncinata (Desv.) Spring (SU), Selaginella heterostachys Baker (SH) and Selaginella remotifolia Spring (SR)] from different harvest time were determined by UV-Vis spectrophotometry and HPLC, respectively. The research aimed to preliminarily confirm the appropriate harvest time of Selaginella plant drugs. Methods: The contents of the total flavonoids and amentoflavone in five Selaginella plant drugs from different harvest time will be determined by UV-Vis spectrophotometry and HPLC, respectively. Results: The proposed methods were rapid and reliable for the quantitative determination of the total flavonoids and amentoflavone in the five plant drugs. The total flavonoids and amentoflavone contents were significantly different in the five plant drugs, and SM showed the highest content in the both contents. The variation tendencies of the total flavonoids and amentoflavone contents from July to October indicated the diversity and complexity of the accumulation of the active ingredients in plants from the same genus. Conclusion: In conclusion, the results provided valuable references for evaluating the quality and preliminarily confirming the appropriate harvest time of Selaginella plant drugs.