Proof that compound-specific stable isotope analysis (CSIA) can be used for both the qualitative and quantitative analyses of biodegradation activities in the subsurface had been lacking. A multitracer test was conducted in the field with ring-deuterated (d5) and completely (d8) deuterium-labeled toluene isotopologues (400 g) as reactive tracers and bromide as a conservative tracer. These compounds were injected into the anoxic zone of a benzene, toluene, ethylbenzene, and xylenes (BTEX) plume down-gradient of the source zone. Qualitative evidence indicated that anaerobic biodegradation of deuterated toluene via the benzylsuccinate synthase pathway had occurred in the aquifer over 4.5 months. Three independent methods were applied to quantify biodegradation of deuterated toluene. Two methods (one: fractionation of toluene-d8 and toluene-d5 using the Rayleigh equation and an appropriate laboratory-derived isotope fractionation factor to calculate microbial decomposition of deuterated toluene isotopologues [CSIA-method], and two: measuring changes in the concentrations of deuterated toluene relative to bromide) gave similar results, implying that the CSIA-method is a reliable tool to quantify biodegradation in contaminated aquifers. Both methods showed a biodegradation of deuterated toluene isotopologues of about 23-29% for the first and 44-51% for the second control plane. A third method involving the mineralization of deuterated toluene isotopologues indicated that parts of deuterium were assimilated into the biomass of toluene degrading microorganisms.