Ultrasound-assisted alkaline pre-treatment (UAAP) and its sequential combination with microwave (SUMAAP) were performed for fractionation of pistachio shell and compared with conventional alkaline pre-treatment (CAP). The pre-treatment conditions were investigated and optimized using Box-Behnken design for maximum hemicellulose and cellulose recoveries. Under optimum conditions, the hemicellulose and cellulose recoveries were 57.67% and 94.05% for UAAP and 66.14% and 90.78% for SUMAAP, while these values were 16.28% and 98.70% after CAP, respectively. The compositional analysis showed that the xylo-oligosaccharides were the main components in liquid phases, while the solid phases were enriched in cellulose. Furthermore, a significant increase in enzymatic hydrolysis yield of pre-treated solids was obtained compared to the raw pistachio shell. Thus, the UAAP was proved to be a reliable and efficient process for pistachio shell pre-treatment and its sequential combination with microwave provide further increase in pre-treatment efficiency while reducing the processing time and alkali requirement. (C) 2020 Elsevier Ltd. All rights reserved.