A growing body of literature shows that Primary Scientific Literature (PSL) is a valuable and useful tool for STEM. Current assessments measuring the success of PSL-based interventions focus mainly on specific scientific content and/or the specific scientific skills students learn. We believe that the underlying motivation students consider while learning to read PSL is equally important to assess. We present 'Motivation in Reading Primary Scientific Literature,' a 19 item novel questionnaire based on student purpose for reading PSL and student efficacy for reading PSL. Exploratory and confirmatory factor analyses (EFA and CFA) were performed on data collected from students enrolled in introductory Biology courses (n = 411 for EFA and n = 325 CFA). Results confirmed 19 items that can be used to measure student motivation in reading PSL through four subscales: (1) expectancy-value, (2) self-efficacy, (3) performance/competence and (4) interest. Preliminary external validity of the Motivation in Reading PSL questionnaire verified differences (p < .05) in students' motivation between one control and two treatment groups after a one-semester PSL-based intervention. These findings suggest that our questionnaire can be used by instructors who implement PSL-based interventions and would like a more comprehensive understanding of how their students approach reading PSL.