AbstractEndocytosis is vital for nutrient uptake and nanomedicine applications, but the biophysics of the pre‐internalization phase remains poorly understood at single‐cell level. This study uses advanced robotic techniques to analyze pre‐internalization adhesion mechanics. MiaPaCa‐2 cells, pancreatic cancer, displayed three interaction phases: rapid lateral displacement, a quasi‐plateau phase, and linear displacement during extraction. Adhesion time is linked to changes in cell mechanics, with MiaPaCa‐2 cells displaying a biphasic uptake process—an initial rapid adhesion phase followed by a strengthening of adhesion high variability in viscoelasticity. In contrast, fibroblasts show a gradual increase in adhesion forces, accompanied by significant rises in stiffness and viscosity. Unlike traditional endocytosis studies, this study focuses on how pathway inhibitors alter initial membrane engagement rather than uptake mechanisms. Clathrin inhibition increased adhesion by 39%, caveolae inhibition by 27%, and microtubule inhibition reduced adhesion by 48%, indicating microtubules' role in adhesion dynamics. Combined inhibition of clathrin, caveolae, and microtubules reduced adhesion by 70%, showing that disrupting multiple pathways severely impairs particle adhesion. Under repeated stress, MiaPaCa‐2 cells soften (≈75% Young's modulus reduction) due to cytoskeletal disruption, while fibroblasts gradually soften (≈71% modulus reduction), highlighting cellular adaptations. These findings provide new insights into the pre‐internalization of particles at the single‐cell level.