As a noninvasive and label-free analytical technique, Raman spectroscopy has been widely used to study the difference between malignant cells and normal cells. Insulinomas are functional -cell tumors of pancreatic islet cells. They exhibit many structural and immunohistochemical features in common with normal pancreatic cells; thus, they are typically difficult to distinguish under the microscope, especially in vivo. We investigated insulinoma and primary rat pancreatic -cell populations using Raman spectroscopy. The details of the optical heterogeneity between these two populations were determined based on different Raman regions primarily involving nucleic acid and protein contents, which are the most distinct cellular contents in these two types of cells. Using principal component analysis–linear discriminant analysis, these two cell types can be readily separated. The results of this work indicate that Raman spectroscopy is a promising tool for the noninvasive and label-free differentiation of insulinoma cells and normal pancreatic cells.