The cultivation of genetically modified (GM) crops has grown rapidly worldwide. This has led to regulatory authorities implementing strict procedures to monitor and verify the presence and abundance of GM varieties in agricultural crops. Immunochromatographic strip tests have been employed for the detection of transgenic proteins expressed in GM crops as rapid, reliable and cost-effective screening tools. In this study, we developed a novel and sensitive strip test assay, based on a sandwich format, for the identification of Cry1Ac and Cry8Ka5 transgenic proteins. We generated two monoclonal antibodies (mAb), namely 1B1 and 5H4, that bind with high specificity to Cry1Ac and Cry8Ka5 proteins. For the development of strip tests, colloidal gold particles were coated with 1B1 mAb and used as the detector reagent. The 5H4 mAb was sprayed at the test line of a nitrocellulose membrane to serve as a capture reagent and anti-species specific antibodies were used at the control line. The strip test developed was capable of detecting 0.06 μg of Cry1Ac and Cry8Ka5 proteins. For validation of the strip test, GM and non-GM cotton leaf samples were employed. The results indicated that the strip test was capable of distinguishing between GM and non-GM cotton samples, offering potential for use as a rapid and cost-effective screening tool for insect-resistant GM crops expressing Cry1Ac and Cry8Ka5 proteins to control lepidopterans and coleopteran pests, respectively.