Control over interparticle distance while creating effective SERS substrates is crucial to exploit an enhanced electromagnetic field. In the present study, phase behaviors of zwitterionic phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and of a 1:1 (w/w) mixture of DPPE with DPPE analogue labeled with Rhodamine B (DPPE-RhB) were used to control optical properties of silver nanocube substrates prepared by the Langmuir monolayer technique. A more homogeneous distribution of the nanocubes was observed in the DPPE/DPPE-RhB mixture, compared to DPPE at all surface pressures due to a more fluid nature of the mixed lipid monolayer. As a result, interparticle coupling and corresponding optical properties can be effectively controlled with the DPPE/DPPE-RhB monolayer. Enhancement of the RhB Raman signal is detected and found to increase exponentially with increasing surface pressure due to the creation of an intense electromagnetic field in interparticle regions. Results of the study show that the phospholipid matrix enables effective control over the interparticle spacing in the monolayer.