Transgenic maize expressing the insecticidal protein Vip3Aa20 is increasingly being adopted in Brazil. In this study, we determined the baseline susceptibility of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) and Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) to Vip3Aa20, as part of an Insect Resistance Management (IRM) program. Diet-overlay bioassays were conducted with neonates exposed to Vip3Aa20 for 7 d. The baseline susceptibility data were obtained for seven field populations of H. armigera and six of H. zea collected from major soybean-, cotton-, and maize-producing areas in Brazil. To validate the diagnostic concentration, 11 field populations of H. zea were tested from 2014 to 2015. The LC50 for H. armigera populations ranged from 2.97 to 8.41 µg Vip3Aa20/cm2 (threefold variation), and for H. zea populations from 0.04 to 0.21 µg Vip3Aa20/cm2 (fivefold variation). The EC50 for H. armigera ranged from 0.099 to 0.455 µg Vip3Aa20/cm2 (fivefold variation), and for H. zea from 0.004 to 0.020 µg Vip3Aa20/cm2 (fivefold variation). H. armigera was more tolerant to Vip3Aa20 protein than was H. zea (≈40- to 75-fold, based on LC50). Based on the LC99 value, the concentration of 6.4 µg Vip3Aa20/cm2 was defined as a diagnostic concentration for susceptibility monitoring in H. zea, and >44 µg Vip3Aa20/cm2 for H. armigera. Our baseline susceptibility data for Vip3Aa20 in H. armigera and H. zea populations will be useful in IRM programs in Brazil.