The species Deguelia utilis and Deguelia rufescens var. urucu, popularly known as "timbó," have been used for many years as rotenone sources in insecticide formulations. In this work, a method was developed and validated using a high-performance liquid chromatography-photodiode array (HPLC-PDA) system, and results were analyzed using hierarchical cluster analysis (HCA). By quantifying the major rotenoids of these species, it was possible to establish a linear relation between them. The ratio between the concentrations of rotenone and deguelin for D. utilis is approximately 1:0.8, respectively, while for D. rufescens var. urucu it is 2:1. These results may help to distinguish these species contributing to their taxonomic identification.