Malaria is a parasitic disease that still prevails in the world and has been widely treated with the use of healing herbs. In this work isolation and extraction strategies have been implemented to get five molecules: 5,8-hydroxy-7-methoxyflavone (1), 6,7-dimethoxy-5,8-dihydroxyflavone (2), 6,7-dimethoxy-5-hydroxyflavone (mosloflavone) (3), 5,6-dihydroxy-7-methoxyflavone (negletine) (4), 5-Hydroxy-7-methoxyflavone (5) and to establish an analytical methodology for the chemical standardization of the different promising extracts from species Piper piedecuestanum. The antiplasmodial potential in vitro was validated on continuous cultures of P. falciparum chloroquine-sensitive strain NF-54 and cytotoxicity was tested in U-937 cell lines. Standardization parameters were checked with desirable acceptance criteria such as linearity, accuracy, precision, limits of detection and quantification. As a result, the best biological responses were achieved in extracts of P. piedecuestanum after 6 h of extraction with dichloromethane, presenting an extraction yield of 3.03 ± 0.306%, an antiplasmodial activity of IC50 = 24.7 ± 3.1 μg.mL−1, a cytotoxicity of CC50 = 17.17 ± 1.19 μg.mL−1, a selectivity index (SI) of 0.7 and a content of marker substances of CM5 = 7.8 ± 0.61, CM1 = 1.9 ± 0.02, CM3 = 4.6 ± 0.39, CM4 = 1.6 ± 0.14 mg.L−1. Compound (1) exhibited promising antiplasmodial activity with an IC50 = 7.33 μg.mL−1 (25.7 μM). The development of this new analytical method and the biological standardization for extracts of P. piedecuestanum enable fulfills the biological specifications and to evaluate future pharmacological studies the therapeutic efficacy in murine models with the most promising.