Once the positive contribution of ultrasound on cellulose acces-sibility was established, the sono-assisted alkali-washing of canebagasse was optimized through a central composite design inwhich three variables (NaOH concentration, temperature, and son-ication power) were studied in different levels.The chemical composition of sono-assisted alkali-washed sub-strates is shown in Table 2, while Table 3 gives the mass recoveryof pretreatment solids and the glucan recovery in relation to theglucan content of the original material, as well as the delignifica-tion rate that was obtained under each pretreatment condition. Thehighest delignification of 66.5% was achieved for sample SA-AWB10(0.1875 g g−1NaOH; 80.23◦C; 105 W), which was shown to have atotal lignin content of 10.26%. Such delignification efficiency was38% higher than that of the sample that was treated with the high-est NaOH concentration (SA-AWB12–0.2926 g g−1NaOH; 55◦C;105 W), suggesting that the temperature was the most significantvariable in the extraction process. Indeed, lower NaOH concentra-tions led to lower delignification levels for experiments that wereperformed at the same temperature and sonication power (e.g., seeSA-AWB11 and SA-AWB12 in Table 3). Therefore, under a sonica-tion power of 105 W, the use of higher temperatures allowed theuse of lower NaOH concentrations, and 0.1875 g g−1(or 0.94% NaOHat 5 wt% TS) was enough to achieve the highest delignification levelunder the conditions used in this study.