High-throughput sequencing based marine microbiome profiling is rapidly expanding and changing how we study the oceans. Although powerful, the technique is not fully quantitative – it only provides taxon counts in relative abundances. In order to address this issue, we presented a method to quantitatively estimate microbial abundances per unit volume of seawater filtered by spiking in known amounts of internal DNA standards to each sample. We validated this method by comparing the calculated abundances to other independent estimates including chemical markers (pigments) and total bacterial cell counts by flow cytometry. The internal standard approach allows us to quantitatively estimate and compare marine microbial community profiles, with important implications for linking environmental microbiomes to quantitative processes such as metabolic and biogeochemical rates.