Author Abstract: Due to the increasing number of authorized events in the European Union, it is crucial for the official laboratories to enforce market control to detect and quantify genetically modified organisms. In this study, an in-house validation of quantitative duplex ddPCR methods was performed involving MON87701, MON87769, MON89788 and CV-127-9 assays with respect to the lectin reference gene. Since the ddPCR methods provide accurate quantification, show less sensitivity to PCR inhibitors and are more suitable for multiplexing compared to the real-time PCR, the optimization of the existing assays was performed with the exception of MON87701, according to the JRC Guidance documents and technical reports. However, some concerns related to practical settings for the quantitative multiplex of ddPCR methods and their validation were encountered; therefore, a general workflow to develop and validate a ddPCR-based method is shown. The obtained data and the validation performance parameters such as specificity, cross-talk, robustness, dynamic range, linearity, the limit of quantification, trueness and precision comply with international recommendations for GMO quantification methods. The duplex ddPCR methods here investigated are equivalent in terms of performance compared to the singleplex real-time PCR methods, showing higher flexibility and cost effectiveness.