A two-dimensional array of gold nanotriangles inscribed onto glass coverslips were optimized for the surfaceenhanced Raman detection of streptavidin/biotin monolayer assemblies. The nanostructures were fabricated by electron beam lithography, and its optical parameters were optimized to be probed under a Raman microscope with a linearly polarized He-Ne laser with an excitation wavelength of λ = 632.8 nm. The platforms were first tested against a monolayer of biotinylated alkanethiols (BAT) functionalized over the gold nanostructure, showing that good-quality spectra could be acquired with a short acquisition time. The supramolecular interaction of streptavidin (strep) with BAT showed subsequent modification of the Raman spectrum that implies a change in the secondary structure of the host biomolecule (streptavidin). Compared to gold surfaces without nanoscale structures, the local enhancement that results from our nanostructured surfaces allows one to detect the vibrational signal of monolayers within a time on the order of seconds and under modest laser intensity, further demonstrating the utility of using plasmonic metallic nanostructures for molecular recognition. © 2011 American Chemical Society.