Background: Genetic blood screening of newborns to detect inborn metabolic disorders has been recognized as a valuable component of neonatal care for decades. Early detection of these conditions has proven to be an effective way to implement interventions that reduce morbidity, death, and related impairments. Aim: To evaluate the effectiveness of a screening toolkit on empowering mothers about neonatal screening tests for metabolic disorders and hearing defects. Design: A quasi-experimental design with pre and post-test was used. Sample: A convenience sample of 152 mothers was included. Instruments: (1): A structured interviewing questionnaire; it includes two parts: (a) Mothers' demographic data. (b) Mothers' awareness about the newborn screening program. (2): Mothers' attitude toward newborn screening program. (3): Mothers' knowledge about metabolic disorders and hearing defects. Results: Mothers had a higher mean score of awareness about newborn screening program, knowledge about metabolic disorders, and hearing defects post-intervention compared to pre-intervention. There was a positive correlation between the total score of mothers' awareness about the newborn screening program, metabolic disorders, and their attitudes about the newborn screening program at the 0.01 level of significance. Conclusion: The study concluded that implementation of a screening toolkit improved mothers' awareness of newborn screening program, and increased knowledge about metabolic disorders and hearing defects. Also, mothers showed a positive attitude toward the newborn screening program. Recommendations: Periodical educational training program for mothers at the maternal and child health centers regarding neonatal screening program, metabolic disorders and hearing defects.