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As genetically modified organisms (GMOs) development is now increasing, detection and determination of their quantitative threshold using reliable methods would be necessary. The goal of this study was to introduce a sensitive method for qualitative and quantitative detection of Roundup-ready soybean samples. For primary screening, semi-quantitative molecular assays have been used for detection of various percentages of transgenic and non-transgenic Roundup-ready soybean samples. Furthermore, an experiment was conducted using the CaMV 35S primers in combinations with soybean lectin-specific primers in two imported samples of soybean seeds. Real-time PCR-based analysis indicated that the amount of GMO material in the seeds and the limit of detection (LOD) obtained for 35S sequence was less than 1%. The sensitivity and accuracy of this method had conformity with the international standards of seed labeling. This is the first report of its type for quantitative detection of a genetically modified material in a commercial seed lot in Iran.