Spider silk fibers of species of the genera Araneus, Gasteracantha, and Linothele sericata were studied. The fibers are composed of axial threads and lateral villi, allowing adhesion to surfaces. Raman spectroscopy was used to determine the surface and internal composition of the threads forming the structure. In the three species, the characteristic amino acid peaks of the spider web were found between 2871 and 2975 cm −1 , which belong to L-glycine, L-alanine, L-glutamine, and L-proline. The threads are composed of a protective layer mainly composed of amides, alanine, and glycine. The fibrils surrounding the axial fibers consist mainly of amide II (1533 cm −1), which allows adhesion between the thread and the surfaces onto which the spider weaves the web. For the genus Linothele sericata, there is a peak on the surface of this spider web located at 2145 cm −1 , which is associated with isonitriles with R-N-C bonds.