AbstractThe glycopeptide vancomycin has an antimicrobial effect by halting the synthesis of the bacterial cell‐wall by binding the D‐Ala‐D‐Ala peptide motif of the cell‐wall precursor Lipid II. This interaction has been extensively investigated using techniques such as XRD and NMR. Here, vancomycin complexed with Ala‐γGlu‐Lys‐D‐Ala‐D‐Ala (the pentapeptide part of Lipid II) is studied by means of Raman and Raman optical activity (ROA) spectroscopy. Where for conventional structural elucidation techniques, the direct study of a drug‐biological target complex is routine, it is not the case for the ROA technique. Through experimental recordings and DFT‐level spectral calculations of the complex, it was found that ROA specifically probes the conformation of vancomycin, even in the presence of the biological target. As such, it could be determined that no noticeable conformational changes are involved when binding Ala‐γGlu‐Lys‐D‐Ala‐D‐Ala. This work showcases the interest of directly examining interacting systems conformationally by means of ROA, potentially complementing and/or confirming findings from other studies, and eventually contributing to a more complete understanding of the structure–activity relationship of a drug compound.