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Background: Responding to stagnating neonatal mortality rates in Ghana, a five-year collaboration called Making Every Baby Count Initiative (MEBCI) was undertaken to improve the quality of newborn care provided around the time of birth. A multi-pronged approach was used to build health worker (HW) capacity in resuscitation, essential newborn care, and infection prevention using a curriculum built on the American Academy of Pediatric's (AAP) Helping Babies Breathe (HBB) and Essential Care for Every Baby (ECEB) modules with an added section on infection prevention (IP).Methods: MEBCI used a training of trainer's approach to train 3688 health workers from district-level facilities in four regions in Ghana between June 2015 and July 2017. Prior to training, HWs familiarized themselves with the learning materials. Concurrently, MEBCI worked to improve enabling environments that would sustain the increased capacity of trained health workers. Knowledge and skills gained were tested using AAP's Knowledge checklist and validated single-scenario Objective Structured Clinical Examinations (OSCEs) tools.Findings: Majority of HWs trained were midwives (58.8%) and came from district-level hospitals (88.4%). Most HWs passed the HBB OSCE (99.9%, 3436/3440). Age of doctors was negatively associated with HBB scores (r = - 0.16, p = 0.0312). Similarly, older midwives had lower HBB scores (r = - 0.33, p value < 0.001). Initiating ventilation within the Golden Minute was challenging for HWs (78.5% passed) across all regions. Overall, the pass rate for ECEB OSCEs was 99.9% in all regions. Classify newborn for further care and communicate plan to family were frequent challenges observed in Volta Region (69.5% and 72.0% pass rate respectively). HWs less than 40 years of age performed significantly better than health workers older than 40 years (p = 0.023). Age of only paediatricians was positively associated with ECEB scores (r = 0.77, p < 0.001) while age of midwives was negatively associated with ECEB scores (r = - 0.08, p < 0.001).Conclusion: MEBCI's integrated HBB-ECEB-IP training resulted in significant mastery of the clinical knowledge and skills of HWs. Harmonization and standardization of the course delivery by trainers and having a core team to ensure training fidelity are essential to maintaining high quality while scaling a program nationally.Funding: Children's Investment Fund Foundation (CIFF). [ABSTRACT FROM AUTHOR]