To harness the quantitative potential of next-generation sequencing for data normalization, spike-in controls are essential. We have engineered three bacterial genomes (Escherichia coli, Staphylococcus aureus, and Clostridium perfringens) to contain a unique synthetic DNA tag that can be detected via 16S rRNA profiling and whole genome sequencing assays. To demonstrate the utility of the spike-in control in microbiome studies, we mixed precise quantities of genomic DNA from the recombinant bacterial strains to create a genomic DNA spike-in standard. This quantified standard was spiked into a known mock community (ATCC® MSA-1000™) containing genomic DNA prepared from 10 different bacterial strains. The resulting data showed that the unique tag of all three bacteria was identifiable and quantifiable by shotgun and 16S rRNA amplicon sequencing using V1/V2, V3/V4, and V4 primers. Spiking these recombinant bacterial genomic DNA at an optimal concentration did not affect microbiome abundance. Further, we demonstrated that the spike-in standard was applicable as an internal control for absolute quantitation. These proof-of-concept experiments support the utility of using a spike-in control with a unique 16S rRNA tag to monitor the full process of a microbiome workflow for both 16S rRNA and shotgun metagenomics assays.