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The reliance on petroleum derivatives and the increasing trend of fuel consumption in the transport sector have brought attention to biofuels as a measure to reduce greenhouse gas (GHG) emissions, enhance energy security and boost economic development. The incentive for utilizing biofuels is their potential to reduce carbon dioxide (CO2) emissions compared to fossil fuels. Biofuels are considered to be carbon-neutral, regarding the direct CO2 emissions associated with their combustion, on the grounds that the carbon dioxide absorbed by the plants through photosynthesis is equivalent to the CO2 released during their combustion. However, a part of the carbon of biofuels may have a fossil origin and therefore, it is responsible for "non-neutral" CO2 emissions. This was recognized by the Intergovernmental Panel on Climate Change (IPCC) in its guidelines for greenhouse gas inventories. Nevertheless, specific guidance of how to estimate fossil CO2 emissions per each type of biofuel, in order to facilitate the work of emission inventory compilers and practitioners, is neither available in the IPCC guidelines nor in the scientific literature. The purpose of this paper is to define a higher tier method to estimate the associated CO2 emissions with the fossil carbon content of biofuels, aiming to cover all possible biofuel types that are mainly used nowadays to replace diesel and gasoline. [ABSTRACT FROM AUTHOR]