Reflective assignments are a powerful tool in undergraduate STEM courses to promote student metacognition and give instructors and researchers a view of that process. We were interested in understanding student challenges in our developmental biology course, so we used reflective assignments to uncover these challenges and also add to our understanding of student metacognition in upper-level courses. We used structural and initial coding and themed student responses. Then, we were able to compare results across two different course formats. We also compared these analyses with student performance on weekly formative assessments and completed item analysis. Our analyses suggest that students struggle the most with application. Notably, student responses did not differ in an asynchronous online class versus a high-flex in-person class. We also surveyed students (n = 162) on their use and perceived value of the metacognitive assignments for further comparison to other studies of upper-level student metacognitive regulation. Results indicate a strong majority of students completed more than half of the assignments, found the assignments beneficial to their learning, and used the opportunity to plan study time. Overall, we find that upper-level college students have accurate metacognitive knowledge, allowing them to identify challenging topics and cognitive skills, and have acquired the metacognitive regulation tools to develop study plans when prompted by a reflective assignment.