This study presents a novel approach to detect genetically modified (GM) plant events that are not covered by common GMO screening methods. It is based on a simplified multiplex assay which merges the event-specific real-time PCR methods for the detection of six GM soybean lines (MON 87701, MON 87708, MON 87769, DP-305423, CV-127 and DAS-68416). The use of two different fluorescent dyes facilitates the subsequent analysis for identification of the GM event. The multiplex PCR method was validated in a collaborative study trial with 16 participating laboratories. Each laboratory received eight samples containing low levels (0.1% or 0.03% m/m) of one or two GM soybean lines and four GM-negative samples. Data of 720 PCR analyses were evaluated and a false-positive rate of 0.3% and a false-negative rate of 3.9% was observed, respectively. The limits of detection (LOD 95%) were calculated based on modelling the probability of detection (POD) and show satisfactory sensitivity and reproducibility for the assay. Furthermore, we discuss the modularity and applicability of event-specific multiplex PCR systems for the detection of GM events that are not covered by screenings.