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This paper describes methodologies for developing efficient surface-enhanced Raman scattering (SERS) substrates such as annealing thin gold films for developing gold nanoislands, fabrication of nanopillars arrays and roughened films by employing focused ion beam (FIB) milling of gold films, as well as overcoating deep-UV-fabricated silicon nanowires with a layer of gold film. Excitation of surface plasmons in these gold nanostructures leads to substantial enhancement in the Raman scattering signal obtained from molecules lying in the vicinity of the nanostructure surface. In this paper, we perform comparative studies of SERS signals from molecules such as p-mercaptobenzoic acid and cresyl fast violet attached to or adsorbed on various gold SERS substrates. It was observed that gold-coated silicon nanowire substrates and annealed gold island substrates provided considerably higher SERS signals as compared to those from the FIB patterned substrates and planar gold films. The SERS substrates developed by the different processes were employed for detection of biological molecules such as dipicolinic acid, an excellent marker for spores of bacteria such as Anthrax.