Newborn screening (NBS) is the largest public health program in the United States, affecting every newborn. The purpose of newborn screening is to identify newborns at risk for selected disorders during the presymptomatic phase, with the hope that early intervention can prevent disease progression. NBS began in the early 1960s following the pioneering work of Robert Guthrie with phenylketonuria. Since then, NBS has expanded, with testing available for more than 50 disorders in most states. Screening tests need to be highly automated, with high sensitivity and specificity to avoid missing patients with disease, and ensuring manageable false-positive rates. Current initiatives in NBS include timeliness to ensure that results of the screen are available by 5 days after birth for a core set of critical conditions. This has resulted in the current recommendation for NBS specimens to be collected at 24 to 48 hours after birth. False-positive rates are higher in the NICU, because of the metabolic instability of sick neonates and the immaturity of premature enzyme systems. The recommended uniform screen panel (RUSP) contains the current list of disorders screened for by most states. Additional disorders continue to be added to the RUSP as medical progress allows previously untreatable disorders to be managed successfully, and thus the need to screen emerges. The costs associated with NBS continue to climb, because despite state-mandated screening, the diagnostic evaluation and treatment of these conditions has no such mandate. This is a particular concern for disorders with annual treatment costs of several hundred thousand dollars.