There are no standard protocols for assessing antibiosis in soybean, Glycine max (L.) (Fabaceae), cultivars to lepidopterans, including tobacco budworm, Chloridea virescens (Fabricius) (Lepidoptera: Noctuidae), an important species that causes damage to soybean crops in several production regions. This study was conducted to assess methodologies to determine the optimal combination of larval density, confinement container size, and plant structure in soybean antibiosis assays to C. virescens. In addition, primary and secondary metabolites in plant structures were analyzed to elucidate potential chemical resistance mechanisms against this lepidopteran. Antibiosis assays to C. virescens in resistant (IAC‐100) and susceptible (BR‐16) soybean cultivars were performed using three larval densities (one, two, and three larvae), two confinement container sizes (150 and 300 ml), and combinations of soybean plant structures (leaves, uncut pods, leaves + uncut pods, cut pods, and leaves + cut pods). Phenol, tannin, fiber, and lignin contents were quantified in the leaves, uncut pods, and grains of the soybean cultivars and related to biological development of C. virescens. Resistance levels in soybean cultivars in antibiosis assays were best differentiated using one larva per 150‐ml confinement container, fed only on soybean leaves. Higher contents of condensed tannins in the leaves of IAC‐100 cultivar may be one of the main chemical mechanisms of resistance to C. virescens. Our study is the first to determine the optimal combination of larval density, confinement container size, and plant structure for antibiosis assays in soybean cultivars to C. virescens. The developed protocol will benefit high‐throughput phenotyping in genetic breeding programs to obtain cultivars resistant to lepidopterans for use in integrated pest management.
