Owing to the labelling requirements of food and feed products containing materials derived from genetically modified organisms, quantitative detection methods have to be developed for this purpose, including the necessary certified reference materials and calibrator standards. To date, for most genetically modified organisms authorized in the European Union, certified reference materials derived from seed powders are being developed. Here, an assessment has been made on the feasibility of using plasmid DNA as an alternative calibrator for the quantitative detection of genetically modified organisms. For this, a dual-target plasmid, designated as pJANUS-02-001, comprising part of a junction region of genetically modified soybean event GTS-40-3-2 and the endogenous soybean-specific lectin gene was constructed. The dynamic range, efficiency and limit of detection for the soybean event GTS-40-3-2 real-time quantitative polymerase chain reaction (Q-PCR) system described by Terry et al. (J AOAC Int 85(4):938-944, 2002) were shown to be similar for in house produced homozygous genomic DNA from leaf tissue of soybean event GTS-40-3-2 and for plasmid pJANUS-02-001 DNA backgrounds. The performance of this real-time Q-PCR system using both types of DNA templates as calibrator standards in quantitative DNA analysis was further assessed in an interlaboratory trial. Statistical analysis and fuzzy-logic-based interpretation were performed on critical method parameters (as defined by the European Network of GMO Laboratories and the Community Reference Laboratory for GM Food and Feed guidelines) and demonstrated that the plasmid pJANUS-02-001 DNA represents a valuable alternative to genomic DNA as a calibrator for the quantification of soybean event GTS-40-3-2 in food and feed products.