Lignocellulosic biomass is a non-edible feedstock that can be used in integrated biorefinery for the production of biochemicals and biofuel. Among lignocellulosic biomass, Cynara cardunculus L. (cardoon) is a promising crop thanks to its low water and fertilizer demand. Organosolv is a chemical treatment that uses numerous organic or aqueous solvent mixtures, and a small amount of acid catalyst, in order to solubilize the lignin and hemicellulose fractions, making the cellulose accessible to hydrolytic enzymes. Lignocellulosic residues of cardoon underwent a two-step treatment process to obtain fermentable glucose. In the first step, the milled biomass was subjected to microwave-assisted extraction using an acidified γ-valerolactone (GVL)/water mixture, yielding a solid cellulose pulp. In the second step, the pre-treated material was hydrolyzed by cellulolytic enzymes to glucose. The first step was optimized by means of a two-level full factorial design. The investigated factors were process temperature, acid catalyst concentration, and GVL/water ratio. A glucose production equal to 30.17 g per 100 g of raw material (89% of the maximum theoretical yield) was achieved after conducting the first step at 150 °C using an acidified water solution (1.96% H2SO4w/w).