[en] An HPLC-UV method for standardization of the methanol-soluble extracts from tubers of Neorautanenia mitis (A.Rich.) Verdc., Leguminosae, harvested during different periods and from different sites, is described. The chemical fingerprint was established with six identified markers using LC-ESI (+)-MS/MS, including rotenone; the total error was used as validation criterion, the accuracy and risk profiles demonstrated the reliability of the method. The study verified that the major degradation product of rotenone in methanol is dehydrorotenone. The detection range of rotenone was between 40 and 400 µg/ml. The collected samples contained 868-5732 µg/g of rotenone. The concentrations of rotenone in the wild samples from the Ngoma site (5167 ± 565 µg/g) were higher than those registered in the samples from the other sites. No significant differences were observed among the remaining sampling sites, and most of the rotenone was located in the inner part of the tubers (2165 ± 1051 µg/g) when compared to that in their peels (961 ± 320 µg/g).