Polyphenols are abundant natural plant micronutrients that commonly contribute to human health due to their anti-inflammatory, antioxidant, antiviral, anti-carcinogenic, anti-aging, anti-allergic, and other biological activities. Their therapeutic benefits mainly depend on the structure, stability, chemical interactions, and absorption, which ultimately affect the bioavailability of these compounds. The bioactivity of polyphenols is evaluated by in vitro and in vivo studies, sometimes yielding inconsistent results due to numerous differences between used models. Among the main differences is the production of reactive oxygen species (ROS) in cultured cell models, potentially leading to misinterpretation of the effects of polyphenolic compounds. Little attention is paid to the polyphenol stability in cell culture medium and the potential generation of artifacts due to their chemical instability. Stability tests of polyphenols are strongly advised to be performed in parallel with cell culture, to help avoid misleading conclusions. This review highlights the existing challenges with cell-based research, focusing on polyphenols' stability in the cell culture media. We also emphasize that new methods analyzing the molecular interactions of compounds with cell culture media supplements are essential to provide a comprehensive understanding of the polyphenols in in vitro models.