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Nowadays, synthetic chemical antidiabetic drugs, besides their therapeutic effects, present adverse effects that could be hard to handle over time. In the last decade, studies reported new alternative molecules with more health benefits and less adverse effects. The goal of this study is to optimize a new antidiabetic formulation using plant flavonoids: Catechin, Epicatechin, and Rutin. They are also a powerful antioxidant and anti-inflammatory molecules. A mixture design experiment will optimize their combination to obtain a new, safe multi-targets antidiabetic formulation making it a powerful combination for the management of diabetes and its complications. To study the variation of blood glucose level in response to the treatment over the time we performed an Oral Glucose Tolerance Test. The blood glucose level variations recorded as responses for the mixture design experiment. We used the molecules at a dose of 10 mg/kg. According to the software analysis, the prediction profiler showed us the optimum combination, and the result was a binary combination between Rutin and Epicatechin (25% and 75%, respectively). This combination prevented hyperglycemia and hypoglycemia, along with the best area under the curve, and after that, we validated it through a repeated oral administration on alloxan-induced diabetic mice for 28 d. Rutin, Catechin, and Epicatechin exhibit a potent antihyperglycemic activity, their synergistic combination validates a new formulation that could be a real candidate to conventional drugs., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020. Published by Elsevier Inc.)