Accurate detection of the microorganisms underlying gut dysbiosis in the patient is critical to initiate the appropriate treatment. However, most clinical microbiology techniques used to detect gut bacteria were developed over a century ago and rely on culture-based approaches that are often laborious, unreliable, and subjective. Further, culturing does not scale well for multiple targets and detects only a minority of the microorganisms in the human gastrointestinal tract. Here we present a clinical test for gut microorganisms based on targeted sequencing of the prokaryotic 16S rRNA gene. We tested 46 clinical prokaryotic targets in the human gut, 28 of which can be identified by a bioinformatics pipeline that includes sequence analysis and taxonomic annotation. Using microbiome samples from a cohort of 897 healthy individuals, we established a reference range defining clinically relevant relative levels for each of the 28 targets. Our assay accurately quantified all 28 targets and correctly reflected 38/38 verification samples of real and synthetic stool material containing known pathogens. Thus, we have established a new test to interrogate microbiome composition and diversity, which will improve patient diagnosis, treatment and monitoring. More broadly, our test will facilitate epidemiological studies of the microbiome as it relates to overall human health and disease.