Aims: The aim of this study was to develop a real-time PCR test for differentiation between Shigella spp. and E. coli, in particular enteroinvasive Escherichia coli (EIEC). Methods and Results: A duplex real-time PCR specific for the genes encoding for beta-glucuronidase (uidA) and lactose permease (lacY) was developed. Ninety-six isolates including 11 EIEC isolates of different serotypes and at least three representatives of each Shigella species were used for selectivity testing. All isolates tested were positive for the uidA gene. Additionally, all E. coli isolates were positive for the lacY gene, whereas no Shigella isolate tested harboured lacY. Conclusions: The duplex real-time PCR assay was found to be simple, rapid, reliable and specific. Significance and Impact of the Study: If possible at all, delineation of so-called inactive EIEC from Shigella spp. is cumbersome. Biochemical and serological methods are limited to specific pheno- and serotypes. This assay clearly simplifies the differentiation of both.