The present work is part of a cross-disciplinary Swedish research project on advanced tailor-made biofuels that aims at identifying drop-in biofuel options for the transport sector that combine excellent combustion properties with sustainable production pathways. The present paper addresses the methodology and primary results of the biofuel production pathway assessment for the diesel fuel alternatives identified within the project. The methodology is illustrated for 2-Ethylhexanol. Three alternative production pathways for 2-Ethylhexanol are analyzed: gasification-based, butanol-based and ethanol-based. The highest biomass to 2-Ethylhexanol conversion (33.9%, lower heating value basis) is achieved for the ethanol-based conversion pathway. The varying spectrum of by-products requires a sophisticated analysis necessary, as addressed in the present work. 2-Ethylhexanol as biofuel cannot outperform conventional biofuels such as e.g. ethanol from a well-to-tank energy performance perspective due to the additional conversion steps necessary. End-use phase benefits such as higher blend-in ratios or reduced pollutant emissions may change the well-to-wheel picture.