In response to the diverse needs of individual students—their unique abilities, interests, learning styles, and cultural backgrounds—K-12 teachers have been using differentiated instruction, supported by research, for decades. While positive results have been shown in K-12 education, the literature to support differentiated instruction in higher education to meet the diverse needs of college students remains inconclusive. To contribute to the literature in this area, this exploratory and qualitative study examined the use of differentiated instruction at a large research institution situated in the southeastern United States with a focus on courses with enrollment of 50 students or more. The participants included 20 instructors teaching large classes within 11 departments and two schools of an academic college that encompasses the arts, humanities, and social and human sciences. The findings suggest that differentiated instruction in large classes at a research university is challenging. Moreover, instructors teaching large classes need a better understanding of differentiated instructional strategies and how to implement them.