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Pretreatment is one of the bottlenecks in the cost and energy-efficient biomass valorization. Deep eutectic solvents are potential candidates for being used to address these challenges. In this work, the deep eutectic solvent composed of choline chloride, and acetic acid was studied for its use in wheat straw fractionation. The pretreated biomass was assessed concerning the lignin and glucan content. Under optimized time and temperature conditions, defined using Doehlert matrix chemometric tool, of 3 h 47 min and 139.6 °C, the processed wheat straw 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. Fractal kinetic analysis of the data showed similar values of k and h for both glucose and xylose reactions between washed and unwashed biomass. This confirmed that biomass washing is an unnecessary step, which in turn opens room for biomass processing intensification. [ABSTRACT FROM AUTHOR]