Bio-oil (pyrolysis oil) is the liquid product of biomass thermochemical conversion. It is a dark, viscous liquid that contains the depolymerization products of hemicellulose, cellulose, and lignin. The physicochemical properties of bio-oils are determined by employing the conventional methods for fuels analysis with proper adaptations. However, the detailed composition of bio-oils in terms of analytes as well as their concentration remains ambiguous and is a challenging task for analytical chemistry. The compounds in the bio-oil range from nonpolar (e.g., hydrocarbons) to highly polar (e.g., phenolics) and from volatile (e.g., organic acids) to nonvolatile (e.g., sugar derivatives), covering a molecular weight (MW) range of about 50-2000 Da. Hence a combination of analytical techniques such as high pressure liquid chromatography, gas chromatography (GC), gel permeation chromatography (GPC), nuclear magnetic resonance spectroscopy (NMR), and Fourier transform infrared spectroscopy (FTIR) are required to determine the bio-oil's composition. Despite the significant breakthroughs of these techniques, they face limitations regarding the sample pretreatment, the incomplete separation and determination of components, and the need of multiple analyses with each method for more complete results. The development of sophisticated, comprehensive, and hyphenated chromatographic and spectrometric techniques such as GC × GC, LC × LC, high-resolution mass spectrometry (HRMS), and 2D-NMR has brought actual advancement in the field of bio-oil analysis. GC × GC and LC × LC have allowed the development of qualitative and quantitative methods for the individual determination of lower MW compounds. However, HRMS and 2D-NMR have facilitated the elucidation of the structure of the higher MW components, offering insight in the effect of pyrolysis conditions on biomass depolymerization and the possibilities for further upgrading of bio-oils.