<jats:title>Abstract</jats:title> <jats:p>As one way to eliminate the issues found in the preceding generation, feedstock exploration in second-generation bioethanol production remains an issue, especially for a tropical country such as Indonesia. From exotic fruit by-products, durian holds a promising perspective that rests on its abundance, superb carbohydrate content and limited usage until now. This work presents the first-ever utilization of durian seeds for sugar production under optimized conditions through alkaline hydrolysis. A simple form of sugar was extracted by varying four parameters, namely substrate loading, NaOH concentration, hydrolysis time and hydrolysis temperature. Response surface methodology based on the Box-Behnken design was employed to outline the most optimum parameter values. Analysis of variance revealed that the quadratic model fit the data appropriately with the order of significance as substrate loading &gt; hydrolysis time &gt; NaOH concentration &gt; hydrolysis temperature. The optimized conditions for reducing sugar yield, as high as 2.140 g/L, corresponded to &lt;50 g/L substrate loading, 0.522 M NaOH, 60 minutes of hydrolysis time and 80oC hydrolysis temperature. The possible ethanol content of 1.094 g/L was also expected under optimized conditions, demonstrating great potential in second-generation bioethanol production.</jats:p> <jats:p>Second-generation bioethanol production from a non-edible feedstock (durian seeds) is optimized by varying key parameters in the alkaline hydrolysis process, showing high yields of fermentable sugars.</jats:p>