<p>The molecular mechanisms and interactions for the complex cancer pathogenesis are still poorly understood. Traditional and conventional endpoint migration assays such as MTT/XTT/CCK-8, transwell migration, clonogenic, and false positives/negatives assays, do not capture the kinetics of the 3D tumor microenvironment. Therefore, there is the need for high-resolution kinetic data on cancer cell invasion, proliferation, migration, cytotoxicity, adhesion, and differentiation, to understand transient and adaptive responses that are often missed by static measurements for highly effective cancer immunotherapeutics. Here, we review the xCELLigence™ Real-Time Cell Analysis (RTCA) system that represents a major advancement in cancer research by enabling non-invasive, label-free, and continuous monitoring of cellular behaviour through impedance-based technology. Over the years, and particularly since 2020, RTCA has been increasingly applied and relied upon across diverse areas of cancer research, including kinase inhibition, gene silencing, metabolic targeting, immunotherapy, nanomedicine, and three-dimensional tumour models. We first analyse the recent advances in the applications of xCELLigence™ RTCA, with a specific emphasis on how impedance-derived kinetic signatures enhance mechanistic understanding of tumor cell behaviour. We then critically discuss quantitative metrics derived from real-time impedance measurements, compare RTCA with conventional endpoint assays, and highlight its translational relevance for drug development and precision oncology. Finally, we outline current limitations and emerging directions that may further consolidate RTCA as a predictive and integrative platform in preclinical cancer research.</p> Graphical abstract <p></p>