Abstract: BACKGROUND: Targeted neonatal echocardiography (Tn-Echo) is a non-invasive examination which may cause pain/discomfort and physiological instability in neonates, but there is little evidence for the same. We conducted this study to evaluate whether targeted neonatal echocardiography causes pain or physiological stress to newborn infants. METHODS: This cross-sectional study was conducted in a tertiary level NICU. Neonates undergoing targeted neonatal echocardiography were enrolled in this study. Pain was assessed using Premature Infant Pain Profile-Revised (PIPP-R) score before, during and after targeted neonatal echocardiography examination. Heart rate, oxygen saturation, perfusion index and blood pressure were also recorded at the same time points. RESULTS: A total of 88 neonates were enrolled. Mean (SD) PIPP-R score during Tn-Echo was 8.18 (2.6) versus 3.60 (1.8) and 4.24 (2.0) before and after respectively (p value < 0.001). Heart rate and respiratory rate were significantly higher during targeted neonatal echocardiography; oxygen saturation and perfusion index were significantly lower during targeted neonatal echocardiography. Preterm infants had higher PIPP-R score compared to term neonates before, during and after the Tn-Echo [8.76 (2.4) versus 6.81 (2.4); p value < 0.001]. CONCLUSION: Targeted neonatal echocardiography causes significant pain/discomfort and physiological instability in neonates.