Detection of GMOs with Real-time PCR is the standard technology for tracing their presence in different sample types ranging from food to plant materials. GM plant seeds are one of the main sources for GM dispersal in the environment, affecting genetic stability and biodiversity. A certified protocol of minimal Real-time PCR reaction volume with low cost is presented, that offers the ability to identify trace amounts of GMOs in seeds, maintaining high specificity, sensitivity, efficiency and robustness (≥95%). The proposed methodology can serve also sampling for environmental monitoring, where handling of increased sample numbers is difficult or/and the sample amount is very low and precious.