The supported lipid bilayer (SLB) is a powerful tool for studying dynamic cell–environment interactions and has been widely used for biosensing applications. Using a reusable microfluidic chip, we present here a strategy to fabricate highly multiplexed SLB and protein arrays for cell signaling research. This approach allows for the rapid patterning of hundreds of highly reproducible and size-tunable SLB arrays with distinct lipid composition and mobility. Using fluorescence microscopy and fluorescence correlation spectroscopy, the lipid mobility is found to play a central role for patterning this membrane assay. Adding protein rings as diffusion barriers extends the accessible mobility range and maintains long-term stability of the hybrid array. Subsequent protein functionalizations on the SLB could be conducted using standard conjugation methods. The utility of the hybrid array for cell signaling experiments is demonstrated by studying the immune NF-κB signaling, whose activity is triggered by the binding of the membrane receptor, toll-like-receptor 4 (TLR 4), to its ligand, lipopolysaccharide (LPS), that is functionalized on the SLB. The patterned array allows cells to adhere and spread on areas without LPS before migrating to interact with membrane-bound LPS to initiate NF-κB activation. Overall, the strategy offers an efficient route to rapidly generate easily controllable and multiplexed molecular arrays that can serve as versatile platforms for biosensing and cell signaling research.