The well-being of the early childhood education (ECE) workforce is of great importance to the field, yet existing measures to capture well-being are varied in conceptual and contextual constructs. There is a need to measure well-being among ECE employees holistically and to be able to compare to other industries. The National Institute for Occupational Safety and Health Worker Well-being Questionnaire (NIOSH WellBQ) is an assessment of workforce well-being through 5 domains that measure the quality of work life, physical and mental health, and conditions outside of work. In this psychometric analysis, we administered the NIOSH WellBQ to 332 ECE staff. We used confirmatory factor analysis to assess the validity of the five NIOSH domains: (1) Work Evaluation and Experience; (2) Workplace Policies and Culture; (3) Workplace Physical Environment and Safety Climate; (4) Health Status; and (5) Home, Community, and Society. Thirteen items were removed from the original survey due to low variability and poor factor loadings to create our final instrument. Model fit of our final model resulted in an AIC of 30780.44, a RMSEA of 0.06, a CFI of 0.78, and an SRMR of 0.07, indicating adequate model fit. Findings indicate that an adapted version of the NIOSH WellBQ has high construct validity and, therefore, can serve as a holistic framework and measurement tool for assessing worker well-being and informing well-being promotion programs among the ECE workforce.