This article is a report of the development of the Parental Stressor Scale: Neonatal Intensive Care Unit (PSS:NICU), which is designed to measure parental perception of stressors arising from the physical and psychosocial environment of the neonatal intensive care unit. Stress theory, literature reviews, expert consultation, and parent interviews guided instrument development and refinement and established the content validity of the instrument. Construct validity is supported by links with theory, correlation with anxiety measures, and factor analytic results. Alpha coefficients support the tool's internal consistency. Three scales were identified: Parental Role Alterations, Sights and Sounds of the Unit, and Infant Behavior and Appearance. Available metrics allow scoring for stress occurrence levels, overall stress levels, and number of stressors experienced. The PSS:NICU can serve as a research or clinical measure to evaluate stressors experienced by parents with infants in a NICU.