Bio-oil or pyrolysis oil - a product of thermochemical decomposition of biomass under oxygen-limited conditions - holds great potential to be a substitute for nonrenewable fossil fuels. However, its high acidity, which is primarily due to the degradation of hemicelluloses, limits its applications. For the evaluation of bio-oil production and treatment, it is essential to accurately measure the acidity of bio-oil. The total acid number (TAN), which is defined as the amount of potassium hydroxide needed to titrate one gram of a sample and has been established as an ASTM method to measure the acidity of petroleum products, has been employed to investigate the acidity of bio-oil. The TAN values of different concentrations of bio-oil components such as standard solutions of acetic acid, propionic acid, vanillic acid, hydroxybenzoic acid, syringic acid, hydroxymethylfurfural, and phenol were analyzed according to the ASTM D664 standard method. This method showed the same linear relationship between the TAN values and the molar concentrations of acetic, propionic, and hydroxybenzoic acids. A different linear relationship was found for vanillic acid, due to the presence of multiple functional groups that can contribute to the TAN value. The influence of the titration solvent on the TAN values has been determined by comparing the TAN values and titration curves obtained from the standard method with results from the TAN analysis in aqueous environment and with equilibrium modeling results. Aqueous bio-oil samples with a known amount of acetic acid added were also analyzed. The additional acetic acid in bio-oil samples caused a proportional increase in the TAN values. The results of this research indicate that the TAN value of a sample with acids acting as monoprotic acids in the titration solvent can be converted to the molar concentration of total acids. For a sample containing acids that act as diprotic and polyprotic acids, however, its TAN value cannot be simply converted to the molar concentration of total acids because these acids have a stronger contribution to the TAN values than the contribution of monoprotic acids. (C) 2017 Elsevier Ltd. All rights reserved.