Bio-oils obtained from the thermochemical conversion of the lignocellulosic biomass are potential sources of renewable materials (bio-fuels, chemicals, …). However, their high chemical complexity and the high amount of oxygenated compounds limit their ready-to-use capacity. Upgrading treatments, such as deoxygenation and/or cracking, have to be applied. In order to assess the most suited and the most efficient upgrading process, it is necessary to obtain an extensive composition description of the bio-oils at all processing stages. In this respect, the non-targeted approach, using Fourier transform high-resolution mass spectrometry combined with different ionization sources, has demonstrated its great abilities to characterize thousands of bio-oil components over a large range of mass and polarity. In this chapter, an exhaustive review of the studies performed in this field will be presented. To define the main challenges related to the bio-oil characterization, some relevant information will be briefly given in regards to the biomass composition and the used processes that ensure its conversion into bio-oils. Additionally, information achieved by “classical” analytical methods will be presented. This will allow demonstrating the contribution of high-resolution Fourier transform mass spectrometry (FTMS) analyses to characterization of bio-oils.