We engineered patterned co-cultures of primary neurons and astrocytes on polyelectrolyte multilayer (PEM) films without the aid of adhesive proteins/ligands to study the oxidative stress mediated by astrocytes on neuronal cells. A number of studies have explored engineering co-culture of neurons and astrocytes predominantly using cell lines rather than primary cells owing to the difficulties involved in attaching primary cells onto synthetic surfaces. To our knowledge this is the first demonstration of patterned co-culture of primary neurons and astrocytes for studying neuronal metabolism. In our study, we used synthetic polymers, namely poly(diallyldimethylammoniumchloride) (PDAC) and sulfonated poly(styrene) (SPS) as the polycation and polyanion, respectively, to build the multilayers. Primary neurons attached and spread preferentially on SPS surfaces, while primary astrocytes attached to both SPS and PDAC surfaces. SPS patterns were formed on PEM surfaces, either by microcontact printing SPS onto PDAC surfaces or vice-versa, to obtain patterns of primary neurons and patterned co-cultures of primary neurons and astrocytes. We further used the patterned co-culture system to study the neuronal response to elevated levels of free fatty acids as compared to the response in separated monoculture by measuring the level of reactive oxygen species (ROS; a widely accepted marker of oxidative stress). The elevation in the ROS levels, was observed to occur earlier in the patterned co-culture system than in the separated monoculture system. The results suggest that this technique may provide a useful tool for engineering neuronal co-culture systems, that may more accurately capture neuronal function and metabolism, and thus could be used to obtain valuable insights into neuronal cell function and perhaps even the pathogenesis of neurodegenerative diseases.