The implementation of novel analytic methodologies in cancer and biomedical research has enabled the quantification of parameters that were previously disregarded only a few decades ago. A notable example of this paradigm shift is the widespread integration of atomic force microscopy (AFM) into biomedical laboratories, significantly advancing our understanding of cancer cell biology and treatment response. AFM allows for the meticulous monitoring of different parameters at the molecular and nanoscale levels, encompassing critical aspects such as cell morphology, roughness, adhesion, stiffness, and elasticity. These parameters can be systematically investigated in correlation with specific cell treatment, providing important insights into morpho-mechanical properties during normal and treated conditions. The resolution of this system holds the potential for its systematic adoption in clinics; its application could produce useful diagnostic information regarding the aggressiveness of cancer and the efficacy of treatment. This review endeavors to analyze the current literature, underscoring the pivotal role of AFM in biomedical research, especially in cancer cases, while also contemplating its prospective application in a clinical context. © 2025 by the authors.; The integration of atomic force microscopy (AFM) into biological facilities, is significantly increasing our understanding of tumors biology and answer to treatments. AFM allows for investigating cell morphology, roughness, adhesion, stiffness, and elasticity in correlation with specific cell treatment, by comparing the morpho-mechanical properties in healthy and treated cells, thereby allowing diagnostic information of the aggressiveness of cancer and the efficacy of any treatment. This review attempts to analyze current literature, highlighting the role of AFM in biomedical research and specifically in cancer case, while also shading some light into novel possible applications in clinics. © 2025 by the authors.