Rapid and inexpensive biosensor technologies allowing real-time analysis of biomolecular and cellular events have become the basis of next-generation cell-based screening techniques. Our work opens up novel opportunities in the application of the high-throughput label-free Epic BenchTop optical biosensor in cell toxicity studies. The Epic technology records integrated cellular responses about changes in cell morphology and dynamic mass redistribution of cellular contents at the 100-150 nm layer above the sensor surface. The aim of the present study was to apply this novel technology to identify the effect of the herbicide Roundup Classic, its co-formulant polyethoxylated tallow amine (POEA), and its active ingredient glyphosate, on MC3T3-E1 cells adhered on the biosensor surface. The half maximal inhibitory concentrations of Roundup Classic, POEA and glyphosate upon 1 h of exposure were found to be 0.024%, 0.021% and 0.163% in serum-containing medium and 0.028%, 0.019% and 0.538% in serum-free conditions, respectively (at concentrations equivalent to the diluted Roundup solution). These results showed a good correlation with parallel end-point assays, demonstrating the outstanding utility of the Epic technique in cytotoxicity screening, allowing not only high-throughput, real-time detection, but also reduced assay run time and cytotoxicity assessment at end-points far before cell death would occur.