Isotope fractionation of fuel oxygenates has been employed as an indicator for monitoring in-situ degradation in the field. For quantification of in-situ degradation, the Rayleigh concept can be applied. The selection of an appropriate isotope enrichment factor (ε) that is representative of the biogeochemical conditions governing the microbial degradation process in the field is crucial for quantification. Therefore, the biogeochemistry of contaminated aquifers has to be taken into account in the development of isotope strategies in assessment and monitoring operations. In addition, controlled microcosms studies are needed to determine the extent of isotope fractionation under different conditions. The simultaneous analysis of carbon and hydrogen isotope composition of fuel oxygenates in a two-dimensional isotope approach opens opportunities for analysis of the predominant degradation process in the field and can be used to select an appropriate fractionation factor. In this contribution we summarise the concept of isotope fractionation of fuel oxygenates to assess in-situ degradation with respect to analytical techniques, recent progress on isotope fractionation in laboratory studies, the concept of two-dimensional isotope analysis, and experience from field studies.