The widespread use of Cry proteins in insecticide formulations and transgenic crops for insect control has led to an increased interest in the environmental fate of these proteins. Although several detection methods are available to monitor the fate of Cry proteins in the environment, enzyme-linked immunosorbent assays (ELISAs) have emerged as the preferred detection method, due to their cost-effectiveness, ease of use, and rapid results. Validation of ELISAs is necessary to ensure accurate measurements of Cry protein concentrations in the environment. Validation methodology has been extensively researched and published for the areas of sensitivity, specificity, accuracy, and precision; however, cross validation of ELISA results has been studied to a lesser extent. This review discusses the use of ELISAs for detection of Cry proteins in environmental samples and validation of ELISAs and introduces cross validation. The state of Cry protein environmental fate research is considered through a critical review of published literature to identify areas where the use of validation protocols can be improved.