Quantitative monitoring of Bacillus thuringiensis (Bt) insecticidal crystal proteins in soil has been hampered by the lack of efficient extraction/detection methods. A novel approach for simple and effective Bt protein extraction was explored by evaluating extraction solutions from invertebrate gut fluids. Marine worm gut fluids were identified as promising for extracting Bt protein from soil. An artificial gut fluid based on these marine worm gut fluids was developed using commercially available chemicals and was evaluated for its ability to extract Bt proteins from soil. On the basis of experiments with Cry1 proteins, the artificial gut fluid in combination with ELISA was highly effective for protein extraction and analysis in a variety of soil types and was well-correlated with bioassay results. Coupling of immunoassay with this extraction method provides, for the first time, an efficient, accurate, and quantitative assay for routine measurement of Bt protein residues in soil.