The human gut is populated by a rich and diverse microbiota that has been described as a human metabolic organ. Its composition has an impact on the health status of the host and could be the target or effector of dietary health effects. The advent of high-throughput sequencing technologies and their integration with advanced analysis methods enables the development of new approaches to characterize the gut microbiota composition and initiates the understanding of its functionality. These technological aspects are not necessarily the limiting factor to successfully identify biological correlations between gut microbiota, diet and health status, as there are other very important aspects. The aim of this article is to address the gut microbiota characterization methodologies, highlight some advantages and challenges, and give our opinion on how critical the sampling and the study design are.