Insulin is a peptide hormone produced by beta cells of pancreatic islets. In type 1 diabetes mellitus, these islets are destroyed by the body’s own immune system, no insulin is produced and the blood glucose level is increased. Nowadays, efforts in combatting type 1 diabetes focus on the transplantation of islets immunoprotected in microspheres made of non-covalently crosslinked hydrogels. The functionality of the encapsulated islets is retained, while the hydrogel matrix allows permeation of the produced insulin into the bloodstream. The applicability of these microspheres has been extensively studied in vivo. However, prior to biological models, a dynamic detection method to monitor the production of insulin and its diffusion through the microspheres is still missing. Herein, we apply the Surface Enhanced Raman Scattering (SERS) technique to detect physiologically relevant concentrations of insulin using planar Ag SERS substrates, while considering their implementation for monitoring insulin diffusion through alginate matrices. Insulin was detected after drying SERS planar substrates in a concentration range of 10-3- 10-12 M. Additionally, we demonstrated the decrease in the deposition time using an alternating electric field. Moreover, the in situ monitoring of the SERS signal from insulin molecules has certain limitations when conducting experiments for SERS substrates submerged in water. As the secretion of insulin and its diffusion across the immunoprotective microspheres is a dynamic process, the development of an adequate detection method is expected to lead to a better understanding of these processes as a function of time, matrix composition, and glucose intake.