H. virescens (tobacco budworm) is one of the three important Lepidoptera pests of cotton that have been continuously monitored since 1995 for Bacillus thuringiensis-resistance. Current methodology to test the susceptibility to this insecticide has been performing bioassays with the progeny of mass-mated moths. Although this approach is valid, the effective gene pool of the tested population cannot be thoroughly assessed due to the complicated reproductive biology of this pest. Utilizing a Bt-resistant colony (Cry1Ac-resistant, known as YHD2) we were able to simulate the necessary conditions to screen for rare Bt-resistance alleles. Since this type of pesticide resistance is recessive, and the likelihood that heterozygous H. virescens present in the field exists, the only method that can detect Bt-resistance is by screening the second generation of this pest segregating heterozygous and homozygous progeny. In order to accurately assess the Bt-resistance frequency of field samples, field-collected H. virescens should be tested individually under the method known as ‘pair-mate' (a field-collected mated with a laboratory-reared H. virescens). Findings show that for an accurate detection method it must involve a minimum number of F1 moths (8-12 pairs) to create the F2 generation. Bioassays with first-instar F2 larvae should be conducted during the second and third moth oviposition day, the period of time when more Bt-resistant larvae are present in a sample. Logistical aspects of this method in order to maximize accuracy and make the best use of resources will be presented.