A quantitative PCR (qPCR) assay with two primers and a TaqMan probe targeting conserved regions of the specific ipaH gene of Shigella species and enteroinvasive Escherichia coli (EIEC) were developed. This qPCR assay was used to identify 206 Shigella strains (including four Shigella species with all serotypes and two provisional Shigella species), 3 EIEC strains, and 113 non-Shigella strains with 100% accuracy. Pure cultures of six Shigella reference strains were used to derive standard curves to determine the detection limit and efficiency of the qPCR method. The ipaH qPCR assay had an equally low detection limit (0.12 to 0.74 CFU per PCR) for the four Shigella species tested. The average qPCR efficiency was 99.29% (95.36 to 103.92%). The detection limit of the qPCR assay tested on 15 varieties of inoculated fresh produce ranged from 0.4 to 16 CFU/100 ml of buffer rinse. This qPCR assay took the variation of wild-type nucleotides into consideration and was used successfully to screen fresh produce. This highly sensitive qPCR assay can be completed within 24 h and has potential use as a screening tool for all four Shigella species and EIEC in food samples.