Acid hydrolysis of levoglucosan and cellobiose as anhydrosugar model compounds was carried out in an autoclave Parr reaction system, using sulphuric acid as catalyst. In addition, acid hydrolysis was carried out using an anhydrosugars mixture from the aqueous fraction of a pyrolysis oil or bio-oil. The bio-oil was obtained from the fast pyrolysis of birch-wood, and the segregated aqueous fraction was found to contain mainly levoglucosan with a concentration of 30 g L-1. Three main hydrolysis parameters including temperature, reaction time, and catalyst to substrate ratios were varied in order to identify their influence towards glucose production. It was found that at hydrolysis conditions of 120 °C, 60 minutes, and a catalyst/substrate ratio of 0.9; glucose yields of 98.55% and 96.56%, and conversion of substrates of 100% and ~92%, were achieved when hydrolysing cellobiose and levoglucosan respectively. An increase in the hydrolysis temperature from 120 °C to 135 °C, resulted in a decrease in the glucose yield and selectivity. Whereas high conversions of substrates (~90%) were maintained for both anhydrosugars. This was attributed to the further dehydration reactions of glucose, possibly yielding HMF or levulinic acid. During the acid hydrolysis of the bio-oil aqueous fraction, a range of hydrolysis conditions suitable to achieve glucose yields higher than 90%, was depicted. It was found that catalyst/substrate molar ratios between 0.17-0.90 and temperatures between 118 °C and 126 °C were suitable conditions to achieve glucose yields ~100% (30 g L-1). Furthermore, glucose concentrations ~117% (35 g L-1) and levoglucosan conversions above 90%, were attained at 135 °C, 20 minutes reaction time and at an estimated catalyst/substrate molar ratio of 0.2 (H2SO4, 0.5 M).