In many anaerobic environments methane (CH₄) is produced by methanogens, with either H₂/CO₂ or acetate (i.e., the methyl group) as precursors, through what are referred to as hydrogenotrophic and acetoclastic methanogenic pathways respectively. Their relative contribution to total CH₄ production can be quantified by determining the stable carbon isotopic fractionation factors for both pathways as well as the isotopic signatures of CO₂, CH₄, and the methyl group in acetate of the sample. The procedures for measuring carbon isotopic fractionation factors of both methanogenic pathways and isotopic composition of these compounds by isotope ratio mass spectrometry are described in this chapter. The results are very helpful in evaluating the activity of the methanogens involved in each methanogenic pathway as well as those of other biological pathways with different fractionation factors.