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Family Centered Care (FCC) in Neonatal Intensive Care Units (NICUs) included family involvement in the care process of newborns and infants. Staff perceptions of FCC may influence clinical practice and management strategies in NICUs, with an impact on quality and humanization of the care. The Family-Centred Care Questionnaire-Revised (FCCQ-R) was adapted for the NICU setting, therefore the FCCQ-R@it-NICU was developed and used for the present study in 32 Italian NICUs. We calculated internal consistency using Cronbach’s alpha correlation between Current and Necessary dimensions of the scale using the Pearson correlation coefficient. Furthermore, we investigated which characteristics could influence staff perceptions of FCC in NICUs. 921 NICU professionals participated in the study. The FCCQ-R@it-NICU revealed good internal consistency (0.96) and good correlation between dimensions (p < 0.05). Statistical and significant differences in Current and Necessary dimensions were found and some demographic characteristics were found predictable on FCC practice. The FCCQ-R@it-NICU is a valid tool to investigate staff perceptions about FCC in NICU settings. Profession, education level and work experience seem to positively influence the perception of what is required for FCC practice within NICUs.