Current World Health Organization recommendations for the diagnosis of soil-transmitted helminths (STH) rely on antiquated microscopy-based techniques that lack both diagnostic sensitivity and specificity. While sufficient for providing rough estimates of infection frequency and intensity within high prevalence settings, these techniques lack the capacity to effectively estimate infection levels following successful intervention efforts, as worm burdens decline and community prevalences decrease. Furthermore, an expanding body of evidence is suggesting that microscopy-based misdiagnosis of infection is likely a larger concern then previously believed. As such, with an increase in programmatic support for transmission interruption and an escalating belief in the possibility of regional eradication, recognition of the need for improved diagnostics is expanding. Such diagnostics are critically important for accurately measuring intervention successes, and for making determinations about where and when interventions can be discontinued allowing for the re-prioritization of resources. Given the stakes and acknowledging this need, recent years have witnessed the growth of molecular diagnostic development efforts, centering primarily upon the creation of real-time PCR-based assays. However, while a variety of assays have been developed, these assays have all utilized suboptimal DNA targets, typically exploiting ribosomal and/or mitochondrial sequences for parasite detection. By coupling next-generation sequencing with bioinformatics-based approaches to the analysis of raw sequencing read data, the selection of optimal DNA-based molecular targets becomes possible. Software, such as RepeatExplorer can be utilized to identify high copy-number, species-specific, repetitive DNA elements within a pathogen. These repeat sequences can then be utilized to design sensitive and specific real-time PCR assays. Utilizing such an approach, we have designed, developed, and extensively validated a panel of assays facilitating the improved detection of the human infecting STH. These assays are proving useful in a number of settings, gaining widespread traction within the operational research community and helping to shape and define future intervention strategies. Furthermore, our assays continue to highlight the inadequacies of alternative diagnostic methods, illustrating the potential challenges and risks for misdiagnosis associated with the use of both microscopy-based diagnostics, and assays targeting less sensitive, less specific genomic regions.