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Cytotoxicity assays are a fundamental tool used in molecular biology, as well as in pharmacy, for drug discovery studies. Most of these assays are based on the measurement of cell viability and proliferation. The reduction of MTT is a widely used colorimetric assay method that determines the viability of cells/cytotoxicity. In recent years, there has been a growing interest in polyphenolic compounds, which exhibit many health-promoting properties and therefore carry enormous therapeutic potential that could be used in the fight against various diseases of civilization (i.e., cancer, atherosclerosis, obesity, and diabetes). The commonly used MTT test may provide incorrect results due to the phenomenon of chemical interaction with plant extracts. More and more assays based on the measurement of intracellular ATP (i.e., CellTiter-Glo) are appearing on the market, and according to many scientists, they are a promising alternative to the tetrazolium salt reduction test. The aim of this study was to compare the utility of the colorimetric MTT assay and the bioluminescent CellTiter-Glo assay for the evaluation of compounds from the polyphenol’s group. Analysis of both methods was performed by comparing the obtained results of the viability of Caco-2 cells treated with a wide range of concentrations of polyphenolic compounds: cichoric acid, caftaric acid, caffeic acid, and cynarin. The results show some discrepancies in the cell survival. Moreover, the overestimation of Caco-2 cell viability using MTT assay confirms the occurrence of interference between MTT assay and analyzed polyphenols. It was confirmed that the CellTiterGlo assay has a higher accuracy of measurement, thus providing more reliable results of cytotoxic analysis of polyphenols compared to the MTT assay. Full article