Neonatal screening based on the “heel prick test” biological matrix is a fundamental test performed in newborns for the screening of genetic, metabolic, and infectious diseases that can lead to health problems or irreversible deficiencies if neglected. The heel prick test is a public health policy adopted in many countries, including Brazil, and is widely recognized as one of the most important procedures in preventive medicine, in order to guarantee comprehensive care and universal access. Thus, the National Neonatal Screening Program (PNTN) was created in 2001, to promote and implement screening actions with a special focus on prevention, early intervention, and monitoring of children whose disease is included in the program's criteria. It is necessary that the health team guide the pregnant woman during the prenatal period, and the puerperal women, as well as their respective companions, on how and where to perform the “heel prick test”, highlighting its purpose to ensure effective awareness. Therefore, the present study aims to analyze the level of knowledge about neonatal screening in pregnant and postpartum women in the public health network. As well as introduce them to the importance of tracking in a timely manner to manage positive cases through monitoring and follow-up during treatment.