Microbial communities are the engines that drive the global biogeochemical cycle of carbon and nitrogen essential for life on Earth. However, microorganisms have evolved as a result of complex interactions with other organisms and environments. Deciphering the metabolism of microorganisms at the community level in nature will be crucial for a better understanding of the mechanisms that lead to the enormous divergence of microbial ecophysiology. Due to the immense number of uncultivated microbial species and the complexity of microbial communities, delineating community metabolism proves a virtually insurmountable hurdle. By tracing the heavy isotope flow of key elements such as carbon and nitrogen, DNA-based stable isotope probing (DNA-SIP) can provide unequivocal evidence for substrate assimilation by microorganisms in complex environments. The essential prerequisite for a successful DNA-SIP is the identification, with confidence, of isotopically enriched 13C-DNA, of which the amount is generally too low to allow the direct measurement of 13C atom percent of nucleic acid. The methodological considerations for obtaining unambiguous DNA highly enriched in heavy isotope are presented with emphasis on next-generation sequencing technology and metagenomics.