Lipid rafts are liquid-ordered domains where specific enzymes and receptors are located. These membrane platforms play crucial roles in a variety of signaling pathways. Thus, alterations in the lipid environment, such as those elicited by oxidative stress, can lead to important functional disruptions in membrane proteins. Cell membrane microarrays has emerged as a powerful methodology for the study of both lipids and membrane proteins, as they preserved native protein structure and lipid environment. Based on this technology, we developed membrane microarrays with different lipid raft preparation from astrocytes and neurons cultured in absence and presence of metabolic and oxidative stress. This raft membrane microarrays were analyzed by MALDI-MS spectrophotometry, identifying a set of lipid species specifically altered in the lipid raft fraction from stress conditions such as PA O-36:3 or PS 34:1. Moreover, lipids noted as PE 40:4 or SM 32:1;O2 could be used as potential markers for astrocytic or neuronal rafts. Furthermore, the enzymatic activity of the NADH oxidoreductase, glyceraldehyde 3-phosphate dehydrogenase and cholinesterase, as well as the density of Sigma-1 and -2 receptors were studied, demonstrating that stress conditions differently affect specific lipid raft proteins, such as Sigma-1 Receptors. In this regard, NADH dehydrogenase activity upon paraquat treatment was reduced while cholinesterase and acetylcholinesterase activities where increased. In summary, we demonstrate the performance of this new microarray technology, adapted for membrane subdomains, to explore changes in lipid composition and enzymatic activities in raft domains from brain cell lines subjected to different stress conditions.