This study was to evaluate the performed painful procedures among all sick neonates cared for at two university hospital neonatal intensive care units (NICUs) in the Republic of Korea. This prospective study was done with all newborns admitted to the NICUs between 1 October and 20 November 2010. Data collection was done with self-administered questionnaire by each nurse for the first 2 weeks of admission and discontinued if the neonate was discharged, transferred or died. The number of painful procedures, according to general characteristics of the neonates, were compared using Mann–Whitney U-test and Kruskal–Wallis test. Oral suctioning was the most frequently performed procedure, followed by tracheal suctioning and nasal suctioning. The number of painful procedures increased as the gestation period became shorter and birthweight decreased. In conclusion, nurses should reappraise the need for painful procedures, in particular, among neonates with a gestation period of ≤ 27 weeks and babies with ≤ 1000 g of birthweight, and execute their performance only if necessary.