Oligomers of β-amyloid (Aβ) contribute to dendritic spine loss and synaptic dysfunction in Alzheimer's disease (AD), yet their underlying molecular mechanisms remain incompletely understood. Rho GTPases, key regulators of cytoskeleton dynamics have been proposed as central mediators in this process. However, most studies to date are inconclusive, and no clear consensus exists regarding their activation dynamics. In this study, we employed new-generation FRET biosensors to quantify, with high spatial and temporal resolution, the activity of RhoA, Rac1, and Cdc42 in primary hippocampal neurons following early and late exposure to Aβ oligomers. Our preliminary data show that, at 5 minutes post-treatment, RhoA and Rac1 activities are significantly elevated in dendrites, whereas Cdc42 activity decreases both 5 and 30 minutes while these effects were not observed at longer times, suggesting a transient and rapid effect. The signaling responses of Rho GTPases within cells are localized in terms of space, time, and duration, with several of them operating simultaneously depending on different stimuli and in each specific subcellular context. To further characterize how Aβ reshapes the interaction networks of RhoA, Rac1, and Cdc42, we will use TurboID proximity labeling to map changes in their interactomes under pathogenic conditions. This approach will enable the identification of novel pathways and molecular assemblies involved in the early synaptic dysfunction events in AD.