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Understanding the global burden of enterotoxigenic E. coli (ETEC) and Shigella diarrhea as well as estimating the cost effectiveness of vaccines to control these two significant pathogens have been hindered by the lack of a diagnostic test that is rapid, simple, sensitive, and can be applied to the endemic countries. We previously developed a simple and rapid assay, Rapid Loop mediated isothermal amplification based Diagnostic Test (RLDT) for the detection of ETEC and Shigella spp. (Shigella). In this study, the RLDT assay was evaluated in comparison with quantitative PCR (qPCR), culture and conventional PCR for the detection of ETEC and Shigella. This validation was performed using previously collected stool samples from endemic countries, from the travelers to the endemic countries, as well as samples from a controlled human infection model study of ETEC. The performance of RLDT from dried stool spots was also validated. RLDT resulted in excellent sensitivity and specificity compared to qPCR (99% and 99.2% respectively) ranging from 92.3 to 100% for the individual toxin genes of ETEC and 100% for Shigella. Culture was less sensitive compared to RLDT. No significant differences were noted in the performance of RLDT using samples from various sources or stool samples from moderate to severe diarrhea or asymptomatic infections. RLDT performed equally well in detection of ETEC and Shigella from the dried stool samples on filter papers. This study established that RLDT is sufficiently sensitive and specific to be used as a simple and rapid diagnostic assay to detect ETEC and Shigella in endemic countries to determine disease burden of these pathogens in the national and subnational levels. This information will be important to guide public health and policy makers to prioritize resources for accelerating the development and introduction of effective preventative and/or treatment interventions against these enteric infections. Author summary: Enterotoxigenic E. coli (ETEC) and Shigella spp (Shigella) causes significant global morbidity and mortality, especially in low-and middle-income countries (LMICs). Since culture methods to detect Shigella are not sensitive, and the methods used to detect ETEC have not been feasible outside of specialized, well-equipped laboratories, the true burden of these pathogens at national and sub-national levels are mostly not available. Morbidity and mortality estimates, for these two pathogens are crucial to assess their relative public health importance in LMICs. We developed a simple and rapid diagnostic assay called the RLDT (Rapid Loop-mediated isothermal amplification based Diagnostic Test) for detection of ETEC and Shigella. In this study we evaluated RLDT compared to other currently available assays using previously collected stool samples. Our data showed that the RLDT assay exhibited high sensitivity and specificity for detection of ETEC and Shigella, with its result available within 50 minutes. The sensitivity of RLDT was higher than culture for these pathogens. We conclude that RLDT could be used as a rapid and simple diagnostic test to determine the burden of ETEC and Shigella in LMICs as well as in clinical vaccine trials of these pathogens. [ABSTRACT FROM AUTHOR]