Measurements of prokaryotic absolute abundance can provide important insights into human gut microbiome biology and correct misinterpretations of relative abundance data. Despite the existence of several relatively well-established methods for making these measurements, most microbiome studies do not report absolute abundance. To enable researchers equipped with standard molecular biology capabilities to incorporate absolute quantification into their microbiome studies, we present a detailed, step-by-step protocol for rigorous and reproducible quantification of prokaryotic concentration in stool samples. We include methods for measuring stool sample moisture content, quantifying the concentration of the 16S rRNA prokaryotic marker gene by qPCR or digital droplet PCR (ddPCR) and analyzing the resulting data. We also highlight and provide strategies to overcome common pitfalls of the quantification method, such as 16S rRNA gene contamination. The final output of this approach is 16S rRNA copies per wet or dry gram of stool. In cases where samples have matched metagenomic sequencing information, data can be converted into absolute concentration of prokaryotes and taxon-specific absolute concentrations. To enable researchers to choose the appropriate method for their specific applications, we also compare and contrast our qPCR and ddPCR methods. In 4 days, ~80 samples can be taken from frozen stool to absolute concentration by using qPCR or ddPCR without the need for resequencing. Overall, this protocol provides a sensitive and straightforward way to measure the absolute concentration of prokaryotes in human gut microbiome samples stored with or without preservative.