Motivation in science learning is believed to be essential for students’ pursuit of college-level studies and lifelong interest in science. Yet, the trend of low levels of motivation in learning science continued in college can be linked to a national concern about low scientific literacy levels and science career aspirations. To diagnose the current status of motivation of college students, it is important to have an instrument that can assess students’ motivation. The purpose of the present study is to examine the level of motivation of college students and establish the validity and reliability of a motivation questionnaire-the Science Motivation Questionnaire II (SMQ II) developed by Glynn et al. (2011)-using the Rasch-Andrich rating scale model. The original instrument consists of 25 items allocated in five sub-factors. Both person separation reliability and item separation reliability were excellent. The item separation index indicated good variability of the items and the five rating scale functioned well. All Infit and Outfit measures in the Rasch analysis demonstrated a lack of unidimensionality of the science motivation construct in the SMQ II, which supports the deletion of two items to satisfy the unidimensional structure.