The genetically modified maize expressing Vip3Aa20 insecticidal protein from Bacillus thuringiensis Berliner is a biotechnological option for the control of Spodoptera frugiperda (J.E. Smith) and Diatraea saccharalis (F.) in Brazil. To support an Insect Resistance Management program, we conducted studies of baseline susceptibility and monitoring of Brazilian populations of S. frugiperda and D. saccharalis to the Vip3Aa20 insecticidal protein. Neonates were exposed to Vip3Aa20 applied on artificial diet surface. Mortality and growth inhibition were assessed after 7 d. All populations were susceptible to Vip3Aa20. The LC₅₀ ranged from 92.38 to 611.65 ng Vip3Aa20/cm² for 16 populations of S. frugiperda (6.6-fold variation), and between 61.18 and 367.86 ng Vip3Aa20/cm² for 6 populations of D. saccharalis (sixfold variation). The EC₅₀ ranged from 21.76 to 70.09 and 48.65 to 163.60 ng Vip3Aa20/cm² for S. frugiperda and D. saccharalis, respectively. There was a low interpopulation variation in susceptibility to Vip3Aa20, which represents the natural geographic variation in the response, and not the variation caused by previous exposure to selection pressure. For these two pests, the diagnostic concentrations of 2,000 and 3,600 ng of Vip3Aa20/cm² caused high mortality. These diagnostic concentrations will be used in resistance monitoring programs in Brazil.