Biomass conversion into solid, liquid and gaseous products by pyrolytic technology is one of the most promising alternative to convert the biomass into useful products and energy. The total characterization of the products from the pyrolysis of biomass is one of the great challenges in this field, mainly due to their molecular complexity. Pyrolysis is a process that causes degradation of biomass in a non-oxidative atmosphere, at relatively high temperatures, producing a solid residue rich in carbon and mineral matter, gases and bio-oil. The yield and properties of the products depend on the nature of the biomass and the type of the pyrolysis process (type of reactor, temperature, gas flow, catalyst). Due to the high molecular complexity of bio-oil, many different technical had been developed to their complete characterization. This chapter describes the principles of the techniques and main application of chromatographic methods (GC, LC, GC × GC, LC × LC, Nano-LC) in the analysis of bio-oils derived from thermo-degradation of biomasses. Especial attention is carried out to two-dimensional techniques that represent the state of the art in terms of separation, sensibility, selectivity and velocity of data acquisition for characterization of complex organic mixtures. For proper use of bio-oil in the chemical industry, it is essential the identification and unambiguous determination of its major constituents. Only then, it is possible to propose a recovery route of some of these components for the development of an industry dedicated to a bio-refinery. For this, chromatographic methods, especially GC × GC/MS, are fundamental because they allow analysis with high sensitivity and accuracy in identifying each constituent of the bio-oil.