The lack of an effective method for inner ear drug delivery is a clinical problem for the prevention and treatment of hearing loss. With technology advances in nanomedicine and the use of hydrogels, more drug delivery options are becoming available. This study tested the feasibility of using a tripartite layer round window membrane (RWM) model to evaluate the effectiveness of a magnetic assisted transport of poly(lactic-co-glycolic acid) (PLGA)/superparamagnetic iron oxide nanoparticles (SPIONs). A RWM model was constructed as a three-cell-layer model with epithelial cells cultured on both sides of a small intestinal submucosal (SIS) matrix with fibroblasts seeded within the matrix. PLGA encapsulated coumarin-6/SPION nanoparticles 100 nm in diameter were formulated by an oil-in-water emulsion/solvent evaporation method and pulled through the RWM model using permanent magnets with a flux density 0.410 T at the pole face. Independent variables such as external magnetic force and exposure time, composition of hyaluronic acid (HA) hydrogel suspending media, and particle characteristics including magnetic susceptibility were studied. Magnetic assisted transport of coumarin-6 labeled magnetic nanoparticles through the RWM inserts increased 2.1-fold in 1 h compared with the controls. HA hydrogel did prevent particle accumulation on the surface of RWM in a magnetic field but also impaired the mobility of these particles. Greater particle susceptibility or stronger external magnetic fields did not significantly improve the transmembrane transport. A RWM model was designed consisting of a SIS membrane and three co-cultured layers of cells, which was structurally and physically similar to the human. PLGA particles (100 nm) with encapsulated SPIONs were transported through this model with the assistance of an external magnet, allowing quantitative evaluation of prospective targeted drug delivery through the RWM via the assistance of a magnetic field.