Microbial processes govern the fate of organic contaminants in aquifers to a major extent. Therefore, the evaluation of in situ biodegradation is essential for the implementation of Natural Attenuation (NA) concepts in groundwater management. Laboratory degradation experiments and biogeochemical approaches are often biased and provide only indirect evidence of in situ degradation potential. Compound-Specific Isotope Analysis (CSIA) is at present among the most promising tools for assessment of the in situ contaminant degradation within aquifers. One- and two-dimensional (2D) CSIA provides qualitative and quantitative information on in situ contaminant transformation; it is applicable for proving in situ degradation and characterizing degradation conditions and reaction mechanisms. However, field application of CSIA is challenging due to a number of influencing factors, namely those affecting the observed isotope fractionation during biodegradation (e.g., non-isotope-fractionating rate-limiting steps, limited bioavailability), potential isotope effects caused by processes other than biodegradation (e.g., sorption, volatilization, diffusion), as well as non-isotope-fractionating physical processes such as dispersion and dilution. This mini-review aims at guiding practical users towards the sound interpretation of CSIA field data for the characterization of in situ contaminant degradation. It focuses on the relevance of various constraints and influencing factors in CSIA field applications and provides advice on when and how to account for these constraints. We first evaluate factors that can influence isotope fractionation during biodegradation, as well as potential isotope-fractionating and non-isotope-fractionating physical processes governing observed isotope fractionation in the field. Finally, the potentials of the CSIA approach for site characterization and the proper ways to account for various constraints are illustrated by means of a comprehensive CSIA field study at the benzene, toluene, ethylbenzene, and xylene (BTEX)-contaminated site Zeitz.