Self-assembled monolayers (SAMs) have been employed for years on plasmonic nanostructured substrates to facilitate surface-enhanced Raman detection of nontraditional molecules. To date, no experiments have been conducted to study the monolayer formation or crystallinity on traditional roughened silver film over nanosphere (AgFON) substrates or to clarify what fundamental properties make a partition layer effective. The work presented herein utilizes surface-enhanced Raman spectroscopy (SERS) band analysis and localized surface plasmon resonance (LSPR) monitoring to examine formation of alkanethiol self-assembled monolayers on AgFONs, with SERS band analysis yielding structural information and LSPR spectral shifts yielding insight about the SAM formation and thickness. Herein, two short chain monolayers and two long chain monolayers were employed to look for SAM thickness and ordering effects over a 72 h time period and under potential control. Overall, the results demonstrate that both the distance a molecule is held from the AgFON surface and the monolayer crystallinity influence which SAM will be an effective partition layer. Also, the ability to tailor monolayer thickness and crystallinity via potential packing has significant advantages over traditional open circuit monolayer formation.