Background: Families with an infant in need of intensive care most often experience a harmful separation after birth. This is due to a division of medical specialties into neonatal care and maternal care. Therefore, a couplet care intervention is implemented for mother-infant dyads in a neonatal intensive care unit. This study protocol provides a comprehensive evaluation of the intervention. The aim is to evaluate the effect and implementation of a complex couplet care intervention to promote zero separation between mother and infant. Methods: The couplet care intervention is a family-centered model of care, where treatment-requiring mother-infant dyads will be admitted together and receive couplet care by neonatal nurses. The study adheres to the framework of the Medical Research Council and will use a mixed methods embedded design comprising a quasi-experimental trial and a qualitative process evaluation. Finally, a health economic evaluation will be conducted to assess the cost-effectiveness of this complex couplet care intervention. Discussion: Separation of mother-infant dyads after birth has an adverse impact on family health and well-being. This study protocol evaluates a complex couplet care intervention. With this study, a first step is taken to help bridge the gap between current practices and a new care model to prevent the separation of mothers and their infants.