Pyrolysis or liquefaction processes can be applied to lignocellulosic biomass to produce a bio-oil which allows the access of green chemicals or sustainable energy. Among the different existing resources, this raw material has the advantage to come from nonfood feedstocks such as agricultural wastes (wood, grass, ...) or dedicated plantations. Whatever the considered bio-oil, the development of high performance analytical techniques is needed to achieve an exhaustive characterization. The use of Fourier transform ion cyclotron resonance mass spectrometry coupled to electrospray ionization (ESI-FT-ICR-MS) has the potential to chemically identify the components of bio-oil at the level of the molecular formula. In this work, we investigated the influence of the sample preparation (use and nature of dopant and ion detection mode) on the development of a robust methodology for lignocellulosic based bio-oil characterization. Commonly used ESI dopants have been studied to increase the ionization yield and the measurement repeatability. We highlighted the dramatic effect of the sample preparation on the global chemical description of the bio-oil, especially the disproportional contribution of the CxHyN1–5Oz species. Moreover, we demonstrated the ability of well-controlled ESI ionization conditions to attain, on the one hand, specific chemical information on the origin (cellulose, hemicellulose, or lignin) of the bio-oil constituents and, on the other hand, the simultaneous description of both its oily and aqueous compounds without a fractionation step.