Flow cytometry is a sensitive and practical technique that can be applied in both basic and clinical research. It allows extracting quantitative and multiparametric valuable information to assist in the study of cell immunophenotyping, morphological complexity, location and/or expression of extra and intracellular molecules involved in metabolic and proliferative pathways, inflammation, viability, and cell death, among others. The parameters are analyzed by labeling antigens of yellow fever virus and/or cells with fluorescent specific monoclonal antibodies or dyes for immunophenotyping and data acquisition using a flow cytometer. In translational research, flow cytometry is a useful tool in tracking and monitoring the etiology, evolution, and outcome of several infectious diseases, such as yellow fever (YF), with the aim of benefiting human health through detection of intracellular viral antigen and vaccine efficacy trials, as well as characterization, quantification, and monitoring of immune cells subpopulations and their biological functions quality. In the last 10 years we have been facing the re-emergence of YF, considered an endemic disease caused by arbovirus in continents including South America. Unfortunately, severe forms of the disease are still associated with increased mortality. Even with the availability of effective vaccines, gaps about understanding the immune pathophysiology and clinical management are still considered a huge challenge for the scientific community. Therefore, such tool has the potential to aggregate in flavivirus setting, being effective in tracking infection in different biological culture systems, animal and human models, as well as in searching for new antiviral drugs and vaccine efficacy.