This research establishes the optimal microwave‐assisted alkali pretreatment (MAP) condition of cassava rhizome (CR) using response surface methodology with Box–Behnken design for enhanced enzymatic hydrolysis glucose yield. The pretreatment parameters included microwave power (300–900 W), irradiation time (5–15 min), and NaOH concentration (3%–7% w/v); and the enzymatic hydrolysis was 24 and 48 hr. Quadratic models were generated and statistical analysis performed to validate the adequacy of the models. The results indicated that the optimal MAP condition was 840 W microwave power, 9 min irradiation time, and 3% w/v NaOH concentration. Under the optimal condition, the predicted and experimental glucose yields were 15.39 and 15.82 g/100 g native cassava rhizome (NCR) for 24 hr hydrolysis, and 16.40 and 16.95 g/100 g NCR for 48 hr hydrolysis, indicating good agreement. In addition, this study examined the effect of MAP on the physical characteristics and morphology of NCR and pretreated CR. The results showed significant structural changes in pretreated CR, indicating that MAP enhanced enzymatic accessibility and glucose yield.