This study aimed at developing and validating an assessment that measures interdisciplinary understanding for the topic carbon cycling. The impetus for this study is the recognition of assessment as “the ‘black hole’ of interdisciplinary education” in K-16 science education (Boix Mansilla, 2005, p. 18). This study followed the construct-modeling framework (Wilson, 2005). A final version of the ISA was developed including 11 multiple-choice (MC) items and eight constructed response (CR) items. 454 students (9th grade to college seniors) were administered the assessment through the Qualtrics online environment. For the CR items scoring rubrics were developed and used to code student responses by a group of evaluators. Agreement between coders was greater than 90%, indicating excellent inter-rater reliability. Item Response Theory (IRT) models, a two Parameter Logistic Model and a Generalized Partial Credit Model, provided evidence of the construct validity of the assessment items. All items reflected unidimensional construct and local independency in the IRT analyses. All except one item were a good fit to the models. The 19 items showed modest internal consistency (Cronbach’s alpha =0.782). The findings suggest that the ISA is a promising and valid tool to assess interdisciplinary understanding in learning carbon cycling.