Healthcare services and the production of healthcare knowledge are increasingly dependent on highly functioning, multidisciplinary teams, requiring greater awareness of individuals’ readiness to collaborate in translational science teams. Yet, there is no comprehensive tool of individual motivations and threats to collaboration that can guide preparation of individuals for work on well-functioning teams. This prospective pilot study evaluated the preliminary psychometric properties of the Motivation Assessment for Team Readiness, Integration, and Collaboration (MATRICx). We examined 55 items of the MATRICx in a sample of 125 faculty, students and researchers, using contemporary psychometric methods (Rasch analysis). We found that the motivator and threat items formed separate constructs relative to collaboration readiness. Further, respondents who identified themselves as inexperienced at working on collaborative projects defined the motivation construct differently from experienced respondents. These results are consistent with differences in strategic alliances described in the literature—for example, inexperienced respondents reflected features of cooperation and coordination, such as concern with sharing information and compatibility of goals. In contrast, the more experienced respondents were concerned with issues that reflected a collective purpose, more typical of collaborative alliances. While these different types of alliances are usually described as representing varying aspects along a continuum, our findings suggest that collaboration might be better thought of as a qualitatively different state than cooperation or coordination. These results need to be replicated in larger samples, but the findings have implications for the development and design of educational interventions that aim to ready scientists and clinicians for greater interdisciplinary work.