Phenolic compounds of Morus alba leaves are bioactive compounds with beneficial properties for human health. Therefore, in this study, an optimization of ultrasonic assisted extraction by Box–Behnken design was used for the first time to optimize factors such as the percentage of ethanol, ratio solvent/sample (v/w) and extraction time to reach the highest phenolic compound amounts (evaluated by HPLC-MS) while also evaluating in vitro antioxidant activity using DPPH, ABTS and FRAP assays. The optimal extraction conditions were 40% ethanol, 1/400 (w/v) and 35 min. Applying these optimal conditions, which were identified and quantified by HPLC-MS, resulted in the extraction of 21 phenolic compounds. According to these results, the main phenolic compounds in Morus alba leaves are the phenolic glycoside and phenolic acid named protocatechuic acid-glucoside and caffeoylquinic. In addition, Morus alba leaf extract contains flavonols such quercetin-3-O-6-acetylglucoside and rutin, which represent more than 7% of its total phenolic content.