The O26 verocytotoxin-producing Escherichia coli (VTEC)-associated outbreak of hemolytic uremic syndrome (HUS) cases in Romania during 2016 showed the need to improve the current methodology of non-O157 VTEC detection and surveillance. An in-house assay based on xTAG Luminex technology was optimized to identify seven of the most relevant diarrheagenic E.coli serogroups (O-specific wzx genes), two convenient VTEC virulence markers (eaeA and ehxA genes), and a species-specific control gene (uidA). Twenty-nine strains previously characterized in terms of serogroup and virulence genes were tested with the optimized protocol and the results were as expected. The ratio of sample signal to background varied from 66.7 (ehxA) to 7.6 (uidA) for positive samples, with a cut-off of 3. Sensitivity varied depending on the target to be amplified from approximately 102 genomic copies to approximately 104 genomic copies per reaction, respectively. The current approach seems an affordable alternative to commercially available assays that can be further exploited to improve existing autochthonous strategies to prevent future VTEC outbreaks.