To identify unauthorised GMOs in food and feed matrices, an integrated approach has recently been developed, targetting pCAMBIA family vectors, abundant in transgenic plants. Their presence is first assessed by qPCR screening and is subsequently confirmed by characterising the transgene flanking regions, using DNA walking. Here, the DNA walking performance has been thoroughly tested for the first time, regarding the targetted DNA quality and quantity. Several assays, on model food matrices mimicking common rice products, have allowed us to determine the limit of detection, as well as the potential effects of food mixture and processing. This detection system allows the identification of transgenic insertions as low as 10 HGEs and was not affected by the presence of untargetted DNA. Moreover, despite the clear impact of food processing on DNA quality, this method was able to cope with degraded DNA. Given its specificity, sensitivity, reliability, applicability and practicability, the proposed approach is a key detection tool, easily implementable in enforcement laboratories.