A wide variety of molecular tools have facilitated a deeper understanding of the structure, composition, and function of microbial communities, and although different molecular markers can be used to characterize and quantify their diversity, the most common molecular marker targeted for survey of prokaryotic cells is 16S rRNA gene. The sequences contained in the nine hypervariable regions of the 16S rRNA gene are unique for specific genera and, therefore, the perfect bullseye to analyze the prokaryotic community or microbiome. Polymerase chain reaction (PCR) assesses the presence, abundance, and diversity of fastidious-growing or unculturable bacteria, and the specificity and efficiency of the primers targeting the 16S rRNA gene are important to reduce the amplification efficiency bias, yielding a higher number of amplicons and hence, sequences. Next-generation sequences (NGSs) brought a revolution in microbial ecology, as its major advantage is the capability to analyze massive quantity of sequences, including sequences from culturable and non-culturable microorganisms at high genomic resolution with high performance and low cost. Quantitative Insight Into Microbial Ecology (QIIME) is an open-source software developed to analyze nucleic acid sequences from a variety of microbial communities improving the microbiome analysis by combining a variety of independent bioinformatics tools. The present chapter proposes a workflow that helps provide a biological sense to the sequences obtained from the Illumina platform, from the demultiplexing step to the alpha and beta diversity analyses.