Bio-oil upgrading through its hydrodeoxygenation (HDO) using sulfided catalysts has attracted significant attention due to its potential to provide advanced biofuels. Although many studies have been undertaken, a detailed understanding of the changes in the chemical composition on the molecular level that would allow the better design of catalysts for bio-oil upgrading is still insufficient. Therefore, we have subjected straw bio-oil and products obtained from its hydrotreatment over a broad range of experimental conditions to a detailed quantitative chemical analysis. Most of the volatile compounds were quantified by GC-MS. Among them, 115 compounds were quantified directly (i.e., using the appropriate standards) and more than 100 indirectly (i.e., based on their structural similarity with corresponding standards). Moreover, the total concentrations of carboxylic acids, carbonyls and phenols were quantified by the Carboxylic Acid Number (CAN), Faix, and Folin-Ciocalteu methods, respectively, to obtain complementary and/or supporting information on the chemical composition to the GC-MS data. The detailed quantification of most volatile compounds in the feed and the products allowed us to create a reactivity order of the oxygen-containing functional groups present and to understand the origin of some of the compounds. Based on the results, the upgrading of straw bio-oil from ablative fast pyrolysis at 340°C and 4 MPa seems to be optimal when evaluating the severity of the reaction conditions and hydrogen consumption, on the one hand, and the products quality, on the other hand. This provides a good starting point for further catalyst development and optimization allowing for the long-term upgrading of the bio-oil for obtaining petroleum-refinery-compatible feedstock.