Background: Neonatal Intensive Care Unit (NICU) nurses with adequate neonatal pain assessment knowledge are crucial in effective neonatal pain management. There is limited research that assessed the knowledge of NICU nurses in Saudi Arabia regarding neonatal pain assessment.Objective: To assess the knowledge of NICU nurses in Saudi Arabia regarding neonatal pain assessment.Design and methods: A cross-sectional design using an online survey was conducted to capture information regarding neonatal pain assessment knowledge from 125 NICU nurses in Saudi Arabia. Knowledge of pain assessment was assessed using a modified version of the knowledge, attitudes, and practice scale. Knowledge scores were classified as high, average, and low.Results: Participants' knowledge regarding neonatal pain assessment was inadequate (Mean = 63/100). The majority of the participants had a low to average level of knowledge (n= 97, 78%).Conclusion: A significant proportion of NICU nurses had inadequate knowledge regarding neonatal pain assessment, which can be improved. Educational interventions are needed to boost these nurses' knowledge regarding neonatal pain assessment.