Deep eutectic solvents are potential candidates for being used in chemistry and chemical engineering as novel solvents. In this work, the deep eutectic solvent composed of choline chloride and acetic acid was studied for its use in wheat straw fractionation, as a greener and low-cost substitute for conventional solvents. The pretreated biomass was assessed concerning the lignin and glucan content. The processed wheat straw, under optimized time and temperature conditions, of 3 h 47 min and 139.6°C, contained as much as 42.5 ± 0.42 wt.% and 38.59 ± 1.26 wt.% of glucan and lignin contents, respectively. The need for biomass washing after the pretreatment with deep eutectic solvents and before the enzymatic hydrolysis step was also evaluated. The obtained enzymatic hydrolysis results, i.e. glucan to glucose yield of 27.13 ± 0.25 vs. 25.73 ± 0.08 for washed or unwashed biomass correspondingly, are equally good substrates. Hence, biomass washing proved to be unnecessary.