The advent of genomics and deep sequencing technologies has facilitated the development of absolute quantification techniques, which offer researchers more objective and precise sequencing outcomes. Unlike traditional relative quantification methods, which provide comparative data, absolute quantification delivers definitive measurements of genes or taxa. This analytical approach mitigates the potential for extraneous influences when comparing disparate samples, thereby reducing analytical errors. The implementation of absolute quantification techniques enhances our comprehension of microbial community structures, ecological dynamics, and their associations with host health or disease conditions. This chapter emphasizes a straightforward and broadly applicable method for genomic quantification, which necessitates the incorporation of a specified amount of internal standard DNA into the samples, eliminating the need for subsequent adjustments during library construction and sequencing. By assessing the proportion of internal standard DNA across various samples, sequencing data can be transformed into absolute quantification metrics. The internal standard method for absolute quantification is versatile and can be effectively utilized across multiple domains, including disease diagnosis, microbial ecology research, the fermentation industry, and environmental monitoring. Overall, absolute quantification methods furnish a more accurate and holistic perspective for microbiome research.