Microbial processing of soil organic matter is a significant driver of C cycling, yet we lack an understanding of what shapes the turnover of this large terrestrial pool. In part, this is due to limited options for accurately identifying the source of C assimilated by microbial communities. Laboratory incubations are the most common method for this; however, they can introduce artifacts due to sample disruption and processing and can take months to produce sufficient CO2 for analysis. We present a biomass extraction method which allows for the direct 14C analysis of microbial biomolecules and compare the results to laboratory incubations. In the upper 50 cm soil depths, the Δ14C from incubations was indistinguishable from that of extracted microbial biomass. Below 50 cm, the Δ14C of the biomass was more depleted than that of the incubations, either due to the stimulation of labile C decomposition in the incubations, the inclusion of biomolecules from non-living cells in the biomass extractions, or differences in C used for assimilation versus respiration. Our results suggest that measurement of Δ14C of microbial biomass extracts can be a useful alternative to soil incubations.