Transgenic cotton has brought about a significant transformation in the management of bollworms. Nevertheless, a considerable challenge has emerged in the form of Pectinophora gossypiella for transgenic cotton. In this research endeavor, we conducted the detection and quantification of Cry1Ac and Cry2Ab protein in field and pot samples of various transgenic cotton cultivars encompassing single, double, and triple gene varieties, employing Bt strips and ELISA methods. The extent of Bt protein production in both field and pot leaf samples across all examined cultivars was assessed at three distinct time points, namely 40, 80, and 120 days after sowing, coinciding with different growth phases (i.e., leaves, squares, bolls, and seeds). The outcomes revealed that heightened levels of Cry1Ac protein were present across all cultivars, whereas elevated levels of Cry2A protein were exclusively observed in the C6 (MNH-1045) cultivar. Bt proteins, namely Cry1Ac and Cry2Ab, exhibited variable concentrations depending on the specific cultivar, growth phase of the crop, and section of the plant. The findings underscored discernible differences in the concentrations of Cry1Ac and Cry2A proteins among all cultivars, while these concentrations also varied across different crop growth periods (i.e., 40, 80, and 120 days) and growth stages (i.e., leaf, square, bolls, and seed). It was discerned that the expression of toxins is contingent upon the developmental stage of the crop and the duration of growth. All of the transgenic cultivars displayed susceptibility to P. gossypiella. However, the triple-gene cultivar exhibited superior resistance. The incidence of P. gossypiella infestations increased in tandem with crop growth. The findings from this investigation are anticipated to provide valuable insights for entomologists and plant breeders, aiding them in devising suitable strategies, such as refuge maintenance or hybrid development, to safeguard transgenic cotton from pink bollworm infestations, thereby contributing to the sustainability of cotton production on a global scale.