p35S promoter and tNOS terminator are the two primary targets for genetically modified organism (GMO) screening. An increasing number of genetic constructions do not contain p35S and tNOS elements; therefore, new screening assays are required. The use of a larger number of screening methods provides a better coverage of the EU-unapproved GMOs and is a cost-effective approach due to the decrease of tests required for identification. In the present study, new real-time PCR screening assays were developed targeting 10 promoter and terminator elements used in genetically modified constructs: pFMV, pNOS, pSSuAra, pTa29, pUbi, pRice actin, t35S, tE9, tOCS, and tg7. Specificity was verified against different plant species, and the limit of detection was determined on plasmid and genomic reference materials. Criteria of performance were successfully tested taking into account the recommendations of international guidelines. It means that these assays can be considered as ready for an inter-laboratory validation.