ABSTRACT Tumor Mutational Burden (TMB) has emerged as a crucial biomarker to guide patient eligibility for immunotherapy. However, whole exome sequencing, the gold-standard method for TMB measurement, remains limited in accessibility due to its high costs, operational complexity, and lengthy processing times. To address these limitations, we investigated whether Ultra-High-Frequency (UHF) technology could serve as a novel approach to assess TMB by analyzing the crossover frequencies or electromagnetic signature (EMS) of cancer cells on a lab-on-a-chip biosensor, integrating microfluidics and dielectrophoresis. In a panel of 12 cancer cell lines with varying TMB levels, we observed that EMS showed an upward shift correlating with higher TMB, particularly in solid tumor cell lines. This finding suggests a potential relationship between TMB and EMS. To further explore this hypothesis, we artificially increased mutation levels by treating cells with the highly mutagenic compound N-ethyl-N-nitrosourea (ENU). Results showed that EMS captured significant TMB variations in ENU-treated cells with enhanced proliferative capacity compared to their parental counterparts. These results underscore the importance of matched control samples for reliable EMS measurements. Altogether, our findings highlight the potential of EMS to detect TMB variations associated with proliferative activity, a key hallmark of cancer cells, thereby enabling a more precise stratification of cancer cells. Highlights We propose a new biosensor to improve patient stratification for ICI eligibility High frequency fields and dielectric spectroscopy can estimate TMB in cancer cells Significant changes in UHF-DEP signatures correlate with varying TMB levels UHF-DEP provides a cheap, rapid and label-free method to predict ICI response Offers an innovative and complementary marker to conventional diagnostic approaches Graphical abstract