Reliable determination of microbial or transgenic Cry toxins is an essential issue in food and feed analyses, and enzyme-linked immunosorbent assays (ELISAs) are the method of choice for quantifying these toxins currently in food and environmental analysis. Internal Quality Control (IQC) is an indispensable method to assess accuracy, precision, and reproducibility of analytical measurements. To assess the utility of the ELISA method, IQC was performed on EnviroLogix Cry1Ab/Cry1Ac QualiPlate ELISA with manufacturer supplied analytical standards. Applicability of negative and positive controls (C− and C+) was examined by Shewhart Control Charts for bias and Control Chart of the Range of Duplicates for precision. Linear regression (up to 5 ng ml−1 Cry1Ab concentration) of the commercial ELISA kit was compared to sigmoid calibration (up to 60 ng ml−1 Cry1Ab concentration). For immunoassay optimization process, possible matrix effects in different liquid and solid vertebrate tissues were examined by determination of the limit of detection values in these matrices.