BACKGROUND: Although motivation of health workers in low- and middle-income countries (LMICs) has become a topic of increasing interest by policy makers and researchers in recent years, many aspects are not well understood to date. This is partly due to a lack of appropriate measurement instruments. This article presents evidence on the construct validity of a psychometric scale developed to measure motivation composition, i.e., the extent to which motivation of different origin within and outside of a person contributes to their overall work motivation. It is theoretically grounded in Self-Determination Theory (SDT). METHODS: We conducted a cross-sectional survey of 1142 nurses in 522 government health facilities in 24 districts of Burkina Faso. We assessed the scale's validity in a confirmatory factor analysis framework, investigating whether the scale measures what it was intended to measure (content, structural, and convergent/discriminant validity) and whether it does so equally well across health worker subgroups (measurement invariance). RESULTS: Our results show that the scale measures a slightly modified version of the SDT continuum of motivation well. Measurements were overall comparable between subgroups, but results indicate that caution is warranted if a comparison of motivation scores between groups is the focus of analysis. CONCLUSIONS: The scale is a valuable addition to the repository of measurement tools for health worker motivation in LMICs. We expect it to prove useful in the quest for a more comprehensive understanding of motivation as well as of the effects and potential side effects of interventions intended to enhance motivation.