INTRODUCTION: Shortly after birth, neonates are exposed to several painful medical procedures, such as newborn metabolic screening, vaccination and venipuncture, without proper management of pain. Unpleasant experiences during the neonatal period are proven to be associated with negative long-term consequences. Non-pharmacological interventions have been studied, although rarely administered and seldom documented. The aim of this systematic review was to assess non-pharmacological approaches to neonatal pain during diagnostic and treatment procedures. EVIDENCE ACQUISITION: Extensive literature research to access randomized controlled trials on non-pharmacological pain management in neonates was performed in MEDLINE (through PubMed), Scopus and Web of Science from October 2011 to September 2021. First analysis included all article titles and abstracts screening to identify relevant studies, and second analysis included a full-text screening of previously selected studies. Eligibility was assessed independently by two authors, and disagreements were resolved by discussion and consensus. In the end, 19 published studies were included, representing a total of 1930 newborns. Main outcome, neonatal pain, was assessed by different neonatal pain evaluation scales. EVIDENCE SYNTHESIS: Non-pharmacological interventions including sucrose/glucose solutions, non-nutritive sucking, breastfeeding, olfactive stimulus, auditory stimulus and sensory stimulus (skin-to-skin care, kangaroo/maternal holding, heat, therapeutic massage, swaddling/facilitated tucking and acupressure) showed decreased behavioral and physiologic pain responses. CONCLUSIONS: Evidence suggests non-pharmacological approaches are safe, effective and can be easily applied in daily practice. There is the need for continued research on non-pharmacological interventions on neonatal pain to help healthcare providers build a tailored pain treatment plan for neonates submitted to procedural pain.