Two anhydrosugar model compounds (cellobiose and levoglucosan), and a mixture of anhydrosugars from the fast-pyrolysis of birch wood were subjected to acid hydrolysis using sulfuric acid as catalyst. The anhydrosugars mixture or bio-oil aqueous fraction was found to contain mainly levoglucosan with a concentration of 30 g L-1. Hydrolysis temperature, reaction time, and catalyst to substrate molar ratios (c/s), were varied to identify their influence for glucose production. At 120 °C, 60 minutes, and 0.9 c/s ratio; glucose yields of 98.55% and 96.56%, and substrate conversions of 100% and ~92%, were achieved when hydrolysing cellobiose and levoglucosan respectively. An increase in the temperature to 135 °C, resulted in a decrease in both glucose yield and selectivity; whereas substrate conversions around 90% were maintained for both anhydrosugars. During the hydrolysis of the bio-oil fraction, a range of conditions to achieve glucose yields above 90%, was depicted. It was found that c/s ratios between 0.17 and 0.90, and temperatures between 118 °C and 126 °C were suitable to achieve glucose yields around 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 and 0.2 estimated c/s ratio.