Mechanobiology is an interdisciplinary field that investigates how living cells and tissues sense, transduce, and respond to mechanical forces and physical cues from their environment, and how these signals regulate biological processes. The increasing recognition of the role of physical forces in shaping cellular responses has challenged the classical biochemical paradigm of cell signaling, which describes cellular behavior as the result of ligand–receptor interactions and downstream molecular cascades. It is now evident that mechanical forces are integral components of these pathways, leading to the so-called mechanochemical paradigm, in which cells integrate chemical and mechanical cues to regulate their function. Within this framework, this thesis applies different biophysical approaches to characterize the mechanical behavior of biological samples, with a particular focus on Atomic Force Microscopy (AFM). AFM was not only used as a measurement tool but also explored as a versatile platform for methodological development, enabling the design of new experimental strategies and correlative approaches with complementary techniques. The common aim of the projects presented in this thesis is to explore new perspectives in the investigation of neurodegenerative diseases. AFM-based Force Clamp Spectroscopy was employed to study the mechanical destabilization induced by the interaction of α-synuclein oligomers, related to Parkinson’s disease, with a biomimetic cytoplasmic membrane model. To investigate cellular mechanotransduction -specifically Piezo1 channel- activity in the presence of pathological oligomers associated with Parkinson’s and Alzheimer’s diseases, a correlative AFM-epifluorescence microscopy setup was developed, allowing calcium fluxes driven by Piezo1 activation to be monitored under pathological conditions. In addition, AFM was used as a rheological tool and combined with Brillouin spectroscopy to investigate altered mechanical pathways in laminopathies, a group of rare neurodegenerative diseases. Finally, AFM force spectroscopy was applied to mechanically characterize different nanocarrier systems for drug delivery, correlating their mechanical properties with their cellular internalization and homing efficiency.