Abstract Opioid use disorder (OUD) has become a national crisis, affecting over 2 million Americans. The number of pregnant women with OUD in the U.S. increased almost 5-fold in the past decade, leading to a similar surge in infants born with neonatal opioid withdrawal syndrome (NOWS), from 1.5 to 8 per 1,000 hospital births (32,000 cases total) between 2004 and 2014. Associated hospital costs have risen by 35%, reaching $532 million in 2014 ($462 million of which was financed by Medicaid). Among neonates prenatally exposed to illicit opioids, 55-94% develop withdrawal symptoms, including respiratory, gastrointestinal, and feeding problems. Infants with severe NOWS often require prolonged hospitalization, and 2-11% develop serious complications, such as seizures and weight loss. Severe cases often require pharmacological intervention to mitigate negative long- term impacts on the infant's neurological development. Per national guidelines, infants born to opioid users are monitored (72-96 hours) in the hospital for signs of NOWS. However, different scales are currently in use to monitor the severity of symptoms, leading to inconsistencies in diagnosis. Moreover, NOWS can present anywhere from birth to 7 days after, sometimes leading to costly readmissions after release. When pharmacologic measures are needed, these delays can lead to an increased risk of infant morbidity. Therefore, there is an urgent need for an accessible, inexpensive, sensitive, and accurate diagnostic tool to support optimal clinical outcomes of infants with NOWS. To meet this need, FYR Diagnostics, alongside the University of New Mexico (UNM) and Texas A&M University (TAMU), will develop a convenient, non-invasive diagnostic assay from umbilical cord samples for early detection of severe NOWS to improve identification of high- and low-risk infants. The assay will be based on two innovations: a) an miRNA signature that identifies infants with severe NOWS; and b) a proprietary patent-pending ultrasensitive DNA Amplification Reaction (UDAR) chemistry that uses a simple primer design to rapidly and directly detect individual miRNAs at low concentrations currently below the limit of detection. In Phase I, FYR will demonstrate the feasibility of this novel quantitative tool by 1) testing the proprietary UDAR chemistry on predictive miRNA biomarkers using umbilical cord plasma samples previously used by UNM/TAMU; and 2) comparing the UDAR- based assay to traditional RT-qPCR-based assays for detection of NOWS miRNA biomarkers to validate assay performance and demonstrate that the UDAR-based assay exhibits equivalent or superior specificity and sensitivity. In Phase II, FYR, UNM, and TAMU will conduct a large-scale clinical study to validate the NOWS early detection tool. The clinical validation study (N=150) will evaluate the analytical and clinical feasibility of the UDAR-based assay, demonstrating that it exhibits comparable performance and predictive utility of NOWS severity acros...