This paper focuses on the determination of the GMO content of maize and soybean samples using real-time PCR, comparing simplex and duplex PCR. The total DNA content of samples was determined by amplifying part of a maize gene encoding a lipid transfer protein, or part of a soybean lectin gene. The transgenic DNA was quantified by amplifying part of the CaMV 35S promoter. The importance of preparation and conservation of standards as well as the relevance of DNA extraction protocol on the variability of results are discussed. For the determination of low GMO content, limitation in the number of copies of the target gene to be amplified is considered. For samples with a theoretical GMO content of 1%, corresponding to the legal threshold for labelling, the value determined by duplex real-time PCR ranged from 0.85% to 1.20%. Both real-time simplex and duplex PCRs allowed identification of GMO free samples without ambiguity.