ABSTRACT A Novel Workflow to Screen for Illicit Drug Exposure in Newborns (RFA-DA-19-019) Neonatal abstinence syndrome (NAS) refers to a spectrum of withdrawal symptoms in newborns who were exposed to illicit or addictive substances in utero. Babies with NAS have higher rates of fetal anomalies (congenital malformations, growth restriction, ischemic placental complications) and perinatal issues (preterm delivery, poor feeding, sleep difficulties, diarrhea, seizures), which together raise the risk for adverse long term outcomes. Rates of NAS have skyrocketed during the last decade and estimates suggest that 5% of mothers use at least one addictive drug during their pregnancy. To address this public health crisis, multiple groups including the American College of Obstetrics and Gynecology and the American Academy of Pediatrics recommend universal screening of substance use in pregnancy using standardized behavioral scoring tools. Unfortunately, such tools are often biased due to subjective scoring or self-reporting errors, and fail to identify babies who did not receive proper prenatal care. Early identification of NAS is essential for referral of affected babies for interventions, including pharmacological treatments and behavioral/social support for the family. Select hospitals in Ohio and Texas are already engaged in universal toxicology based newborn screening (NBS) for NAS, and several state public health programs are evaluating feasibility to incorporate NAS screening into their standard NBS panels. While toxicology NBS for NAS is expanding in the U.S., several factors currently restrict the growth of such programs. These include: limited sensitivity and short history of exposure with existing tests for urine and blood samples, challenging methodologies for meconium sample processing, limited testing options for meconium samples, and long turnaround times for the few existing meconium tests. The potential consequences of delayed or otherwise insufficient NAS screening include increased errors and/or delays in pharmacologic treatments, high costs associated with unnecessary hospitalization, and increased risk for misdiagnosis. To combat these challenges, we propose a novel workflow that will enable rapid toxicology screening of urine or meconium samples in the hospital. Our system will pair a simple sample preparation protocol with a high sensitivity panel of homogeneous enzyme immunoassays recognizing five common classes of drugs: fentanyl, morphine, amphetamine/methamphetamine, cocaine, and benzodiazepines. The tests will be automated on an innovative digital microfluidic analyzer with a total time to result, including sample processing time, of under 2 hours. The product of this research meets the goals of RFA-DA-19-019 to develop ?innovative methods to identify and treat newborns exposed to opioids? and will furthermore support the goals of the American Academy of Pediatrics to expand high sensitivity NAS screening. The potential benefits from implementation of our protocol include reduced length of hospitalization for unaffected newborns, accelerated time to confirmatory results, faster resolution of acute withdrawal symptoms, and improved referral to family/maternal support services.