The Raman optical activity (ROA) spectroscopic technique has been applied in the past to many biologically relevant systems including peptides, proteins, sugars, and even viruses. However, theoretical interpretation of the spectra relies on lengthy quantum-chemical computations, which are difficult to extend to larger molecules. In the present study, ROA and Raman spectra of insulin under a range of various conditions were measured and interpreted with the aid of the Cartesian-coordinate tensor transfer (CCT) method. The CCT methodology yielded spectra of insulin monomer and dimer of nearly ab initio quality, while at the same time reproducing the experimental data very well. The link between the spectra and the protein structure could thus be studied in detail. Spectral contributions from the peptide backbone and the amino acid side chains were calculated. Likewise, specific intensity features originating from the α-helical, coil, β-sheet, and 3(10)-helical parts of the protein could be deciphered. The assignment of the Raman and ROA bands to intrinsic molecular coordinates as based on the harmonic force field calculation revealed their origin and degree of locality. Alternatively, the relation of the structural flexibility of insulin to the inhomogeneous broadening of spectral bands was studied by a combination of CCT and molecular dynamics (MD). The present study confirms the sensitivity of the ROA technique to some subtle static and dynamic changes in molecular geometry, and many previous ad hoc or semiempirical spectral-structure assignments could be verified. On the other hand, a limitation in longer-range tertiary structure sensitivity was revealed. Unlike for smaller molecules with approximately equal contributions of the electric dipole (α), quadrupole (A), and magnetic dipole (G') polarizabilities, only the electric dipolar polarization (α) interactions seem to dominate in the protein ROA signal. The simulations concern the largest molecule for which such spectra were interpreted by a priori procedures and significantly enhance protein folding studies undertaken by this technique.