The widespread use of Bacillus thuringiensis (Bt)- based biopesticides in organic farming presents challenges for food diagnostics due to the genetic similarity between Bt and the pathogenic Bacillus cereus as well as the lack of data on the insecticidal protein concentrations in treated food samples. To address these issues, a liquid-chromatography – high-resolution tandem mass spectrometry – parallel reaction monitoring (LC-HR-MS/MS-PRM) method was developed for the identification and absolute quantification of insecticidal crystal proteins from Bt subspecies kurstaki, aizawai, and israelensis. Marker peptides were selected based on untargeted data-independent acquisition (DIA) analyses and validated for subspecies differentiation and quantification using stable isotope-labeled (SIL) standards. The method demonstrated high sensitivity (LOD and LOQ in the ng/g range), repeatability precision, and selectivity across various sample matrices. Application to basil plants treated with commercial Bt products confirmed accurate subspecies identification and quantification of crystal protein expression under realistic conditions. This approach enables reliable verification and differentiation of Bt subspecies, supports product quality control, and contributes to environmental safety assessments by providing quantitative data on Bt crystal proteins in food samples. By verifying the presence of declared subspecies and quantifying insecticidal protein content, this method contributes to regulatory compliance and risk evaluation. It offers a robust analytical tool for food safety diagnostics and biopesticide monitoring, addressing key limitations of current approaches.