Purpose: Many instructional strategies for cooperative learning have been used in medical education, such as team-based education, inter-professional education, and problem-based learning. However, useful assessment tools for investigating students' readiness for cooperative learning are lacking. Therefore, the present study developed the Positive Social Interdependence Readiness Scale (PSIRS) questionnaire based on the cooperative learning theory. This paper presents the development and validation of the PSIRS for use with undergraduate health care students in the context of cooperative learning. Methods: A version of the PSIRS questionnaire was administered to 207 health care undergraduates who participated in cooperative learning activities under three models at Taipei Medical University. Results: Three factors, measured by 39 survey statements, were identified from the statistical analysis of the survey data, namely cooperative learning appreciation, the psychological characteristics of Positive Social Interdependence, and students' confidence in teamwork effectiveness. The internal consistency measure was 0.983. Analysis of variance demonstrated some key differences in the three factors among courses. Conclusions: The validated PSIRS questionnaire was proven to be applicable and able to differentiate between student subgroups. More applications and factor influencing PSIRS inquiry are suggested in the future.