Pretreatment is a necessary step when lignocellulosic biomass is to be converted to simple sugars / however single-stage pretreatment is often insufficient to guarantee full availability of polymeric sugars from raw material to hydrolyzing enzymes. In this work, the two-stage pretreatment with use of acid (H2SO4, HNO3) and alkali (NaOH) was applied in order to increase the susceptibility of Jerusalem artichoke stalks (JAS) and oat straw (OS) biomass on the enzymatic attack. The effect of the concentration of reagents (2% and 5% w/v) and the order of acid and alkali sequence on the composition of remaining solids and the efficiency of enzymatic hydrolysis was evaluated. It was found that after combined pretreatment process, due to the removal of hemicellulose and lignin, the content of cellulose in pretreated biomass increased to a large extent, reaching almost 90% d.m. and 95% d.m., in the case of JAS and OS, respectively. The enzymatic hydrolysis of solids remaining after pretreatment resulted in the formation of up to 45 g/L of glucose, for both JAS and OS. The highest glucose yield was achieved after pretreatment with 5% nitric acid followed by NaOH, and 90.6% and 97.6% of efficiency were obtained, respectively for JAS and OS.