Universal newborn screening for human immunodeficiency virus (HIV) has been mandatory in New York State (NYS) since 1997. The goal of screening is to prevent mother-to-child transmission (MTCT) by using approaches including antiretroviral therapy (ART) in the newborn and lactating mother. Screening is performed using an enzyme-linked immunosorbent assay that detects maternal antibodies in dried blood spots from newborns. From January 2011 through November 2018 and from October 2022 through December 2024, the NYS Newborn Screening Program used the HIV-1 MicroELISA System to screen for anti-HIV-1 antibodies in these samples. Newborns who screened positive were referred to an infectious disease specialist for follow-up. During the period of this study, approximately 2.3 million newborns were screened for HIV-1 antibodies. Amongst 4018 newborns referred for follow-up, MTCT was confirmed in 16 newborns (0.4%), 3930 were uninfected (97.8%), 53 were lost to follow-up or were deceased (1.3%), and 19 newborns were considered to have false-positive HIV results (0.5%). MTCT occurred in one in 144,162 newborns screened during the study period, while one in 577 infants were considered to be at risk of vertical HIV transmission. The numbers of newborns who screened positive for HIV antibodies and were therefore at risk of MTCT as well as MTCT have both fallen from 2011 to 2024. NYS MTCT has been below 1% since 2015. To further reduce transmission risk, factors that contribute to loss to follow-up of infants exposed to HIV should be investigated.