Biofuel produced from biomass pyrolysis is a good example of a highly complex mixture. Detailed understanding of its composition is a prerequisite for optimizing transformation processes and further upgrading conditions. The major challenge in understanding the composition of biofuel derived from biomass is the wide range of compounds with high diversity in polarity and abundance that can be present. In this work, a comprehensive analysis using mass spectrometry is reported. Different operation conditions are studied by utilizing multiple ionization methods (positive mode atmospheric pressure photo ionization (APPI), atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) and negative mode ESI) and applying different resolving power set-ups (120 k, 240 k, 480 k and 960 k) and scan techniques (full scan and spectral stitching method) to study the complexity of a pyrolysis biofuel. Using a mass resolution of 960 k and the spectral stitching scan technique gives a total of 21,703 assigned compositions for one ionization technique alone. The number of total compositions is significantly expanded by the combination of different ionization methods.