Monsanto has commercialized corn varieties protected against insect feeding damage by producing the Bacillus thuringiensis proteins Cry3Bb1 and Cry1Ab. Activity of the Cry3Bb1 protein is restricted to the order Coleoptera and most evident within the family Chrysomelidae, whereas, the activity of Cry1Ab protein is restricted to the order Lepidoptera. To support environmental assessments and Monsanto’s analytical protein standards program, bioassay procedures utilizing diet-incorporation methodology have been developed and validated for detection and quantification of biological activity. Colorado potato beetle (CPB; Leptinotarsa decemlineata) and corn earworm (CEW; Helicoverpa zea) were chosen as the model organisms for these assays because of their commercial availability and ease of rearing. Additionally, CPB is highly sensitive to the Cry3 class proteins and CEW is relatively sensitive to the Cry1 class proteins. A comparison of CPB 7-day EC50 and LC50 values for the Cry3Bb1 protein demonstrated similar sensitivity for both endpoints, however there was lower inter-assay variability with LC50 values. This contrasts with the CEW assay for the Cry1Ab protein, where 7-day EC50 values are orders of magnitude lower than LC50 values. LC50 values from independent assays conducted over the past 4 years with the purified Cry3Bb1 protein and Cry1Ab protein incorporated into an artificial diet have ranged from approximately 0.30 to 1.0 ug /ml diet and 0.0012 to 0.0043 ug/ml diet, respectively. The results from these historical assays were used to develop acceptance criteria that were subsequently validated. A synopsis of the bioassay methodology and the approach used to validate these assays will be presented.