In Vitro Methods for Assessing Polyphenol-Based Inhibition of the Diabetes Key Target α-Glucosidase: A Critical Analysis of Methodological Variability

α-Glucosidase is a key therapeutic target in the management of type 2 diabetes, as its inhibition delays glucose absorption into the bloodstream and thus attenuates postprandial hyperglycemia. Although several clinically approved antidiabetic agents function as α-glucosidase inhibitors, their clinical use is often constrained by gastrointestinal adverse effects. These side effects have driven efforts to identify novel α-glucosidase inhibitors with improved therapeutic index. Nonetheless, this increasing interest in the search for new compounds has been hampered by methodological difficulties associated with α-glucosidase inhibitor screening assays. Despite the considerable attention given to natural sources, especially polyphenolic compounds, the substantial differences in experimental setups across studies can compromise the reproducibility and comparability of results reported for the same molecules. This review aims to identify and summarize the methodologies and experimental conditions used to evaluate the inhibitory activity of polyphenols against α-glucosidase. A comprehensive analysis of 102 studies was conducted, focusing on key experimental parameters, including enzyme source and substrate type with their respective concentrations, temperature, pH, buffer, and incubation time employed in the assays. By pinpointing methodological inconsistencies and drawing attention to recurring best practices, this work underscores the importance of increased standardization to ensure reliable cross-study comparisons and to advance the rational development of polyphenol-based α-glucosidase inhibitors.