Background <p>Family-centered care (FCC) has become the gold standard of intensive care practice, especially in the neonatal intensive care units (NICUs), due to its positive impact on family and neonatal health outcomes. However, there are few validated instruments specifically designed to assess FCC practices in low-resource NICU settings. This study adopted the Family Systems Theory (FST) as a theoretical framework to develop and validate an instrument for assessing the implementation of FCC in low-resource NICUs.</p> Methods <p>This study was an instrument development and validation in a three-phased mixed-method triangulation design. Phase I consisted of a scoping review, 24 interviews, and 12 focus group discussions to generate a 40-item draft. Phase II involved piloting (<i>n</i> = 30) the drafted instrument for two quantitative surveys (<i>n</i> = 446) among family (<i>n</i> = 346) and clinicians (<i>n</i> = 100) for item reduction with exploratory and confirmatory factor analysis. Phase III entailed instrument validation by estimating validity and reliability through a two-round modified Delphi study with experts (<i>n</i> = 25). The study conforms to the STROBE checklist of instrument development and validation.</p> Results <p>Factor analysis produced a validated 20-item instrument, the Family Acceptance (4 items), Education (6 items), Environmental Design (6 items), and Sufficient Resources (4 items) (FEES) instrument. The instrument has acceptable validity and reliability; Kaiser-Meyer-Olkin (KMO) was 0.943, CFI = 0.969, TLI = 0.995, ω = 0.955, Cronbach’s alpha = 0.949, and average Intra-Class Correlation of 0.951. The expert validation of the item-level content validity index (I-CVI) ranged from 0.88 to 0.96, and the scale-level content validity index (S-CVI/Ave) is 0.924.</p> Conclusions <p>An instrument has been developed and validated, which demonstrates acceptable psychometric properties, potentially contributing to knowledge and practice in paediatric nursing. The FEES instrument provides a reliable tool to assess FCC practices in NICUs in neonatal care, which could result in the adjustment of nursing interventions to improve healthcare outcomes for critically ill infants and their families. The FEES instrument has further illuminated the importance of FCC in healthcare, especially NICU practice. It could guide the design of future research methodology, instrument development, and advanced statistical analysis to enhance practice and education in nursing science.</p>