BACKGROUND AND OBJECTIVES:The importance of recognizing how the nurse acts in the identification, assessment, treatment, and relief of pain since the newborn is not able to express it orally. The objective of this study was to identify the practices and challenges of the nurse in the assessment and treatment of pain in newborns of a neonatal intensive care unit of a reference hospital in the western region of the Brazilian Amazon. METHODS: A descriptive, field-based, qualitative approach was chosen. Eleven nurses participated in the study, starting with an interview, following a semi-structured script. The content analysis method was used for data analysis. RESULTS: The participants do not recognize the term "fifth vital sign" in the evaluation of pain, but they believe in the ability of the newborn to feel pain, identifying it mainly by the cry and facial expression. Among the procedures considered most painful are punctures and aspiration. Nurturing and non-nutritive sucking are among the actions most indicated to prevent and relieve pain. CONCLUSION: We suggest the implementation of protocols, standards, and routines for the assessment and quantification of pain since when not assessed, it can prolong the hospitalization time. The perception of the nurse in the identification of pain signs in a systematized manner, promotes quality and humanized care, and reduces injuries.