Extracellular vesicles (EVs) are important mediators of intercellular communication in several physiopathological conditions. Oxidative stress alters EVs release and cargo composition depending on the cell type and stimulus. Recently, most of the EVs studies have focused on the characterization of their cargo, rather than on the morphological features (i.e., size distribution, shape, and localization on the cell surface). Due to their high heterogeneity, to fully characterize EVs both the functional and morphological characterization are required. Atomic force microscopy (AFM), introduced for cell morphological studies at the nanoscale, represents a promising method to characterize in detail EVs morphology, dynamics along the cell surface, and its variations reflecting the cell physiological status. In the present study, untreated or H2O2-treated wild-type and SOD1-G93A SH-SY5Y cells have been compared performing a transmission electron microscopy (TEM) and AFM morpho-quantitative analysis of budding and released vesicles. Intriguingly, our analysis revealed a differential EVs profiling, with an opposite behavior and implying different cell areas between WT and SOD1-G93A cells, on both physiological conditions and after H2O2 exposure. Our results empower the relationship between the morphological features and functional role, further proving the efficacy of EM/AFM in giving an overview of the cell physiology related to EVs trafficking.