Background: It was estimated that 15 million preterm neonates were born worldwide each year, with over one million of those neonates dying as a result of prematurity. The aim of developmental supportive care is to create a womb out of womb which improve comfort of the preterm neonates in the Neonatal Intensive Care Unit. Aim of this study was to assess the effect of simulative heartbeat nest on improving physiological parameters, comfort and pain of preterm neonates at neonatal intensive care unit. Research design: A quasi experimental design was utilized in this study. Setting: This study was conducted at neonatal intensive care unit in Specialized Pediatric Hospital at Benha City. Subjects: Purposive sample of preterm neonates (60) at neonatal intensive care was involved. Tools of data collection Tool (I): A structured interviewing questionnaire: It included three parts: Part (1): Personal characteristics of the studied preterm neonates. Part (2): Physical parameters of preterm neonates. Part (3): Physiological measurements of the studied preterm neonates. Tool (II): Premature baby comfort scale. Tool (III): Premature baby pain profile (PIPP-R).Results: There were a highly statistically significant difference (P-value <0.001) between premature neonates in study/control group at the pre/post using of simulative heartbeat nest concerning their comfort, physiological parameters and pain score. Conclusion: It was concluded that using of simulative heartbeat nest was a highly effective method to improve physiological parameters, comfort and pain of preterm neonates. Recommendations: Implementing strategies of developmentally supportive care like nesting that mimic the intrauterine environment in the NICU that improves preterm neonate's state of alertness, posture, comfort and stable physiological parameters.