A PCR-based DNA walking analysis was performed on a protease product suspected to contain a new unauthorized genetically modified microorganism (GMM). Though the characterization of unnatural associations of sequences between the pUB110 shuttle vector and a Bacillus amyloliquefaciens gene coding for a protease, the presence of the GMM was shown. Based on these sequences of interest, a real-time PCR marker was developed to target specifically the newly discovered GMM, namely GMM protease2. The performance of the real-time PCR marker was assessed in terms of specificity and sensitivity. The applicability of the real-time PCR GMM protease2 marker was also demonstrated on microbial fermentation products. To confirm its use by other GMO enforcement laboratories, the transferability of the in-house validated real-time PCR marker was demonstrated by assays performed by an external laboratory.