© 2019 Shaw et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Trichomonas vaginalis is the most common curable sexually transmitted infection (STI) worldwide. Although predominately asymptomatic, the disease spectrum of trichomoniasis in women is characterized primarily by signs and symptoms of vaginitis, including purulent discharge and localized vulvar pruritus and erythema. Several FDA-cleared nucleic acid amplification tests (NAATs) are available for the diagnosis of T. vaginalis infections, but laboratory developed tests (LDTs) are widely utilized and cost-effective solutions in both the research and clinical diagnostic settings. LDT diagnosis of T. vaginalis is particularly appealing since it can be performed using remnant specimens collected for other STI testing. Using a LDT implemented as part of this study, T. vaginalis was detected in 7% of participating Louisiana women (14/199). The mean T. vaginalis organism burden was 1.0x106 ± 4.5x105 organisms per mL of ThinPrep PreservCyt. Using DNA eluates obtained after HPV testing on the cobas 4800 system, the T. vaginalis LDT was characterized by excellent intra- and interassay reproducibility (coefficient of variation values all <3.5%). Compared with two commercially available NAATs from TIB MOLBIOL, the sensitivity and specificity of the LDT was 92.9 and 99.5%, respectively. Collectively, this study details the diagnostic and quantitative utility of a LDT for T. vaginalis. When applied in the clinical research setting, we confirmed the high prevalence of T. vaginalis, but also observed extraordinarily high organism burdens in the cervix. These findings highlight the unique host-pathogen relationship of T. vaginalis with lower reproductive tract tissues, and substantiate the need for continued investigation of this highly prevalent STI.