Infectious diseases are a major cause of mortality in the world and, among them, dengue is considered the main human arbovirus. No effective vaccines or antiviral drugs are available for this illness, and it is estimated that 2.5 billion people live at risk, leading to millions of dengue cases annually. The most common method for dengue virus (DENV) quantitation is the plaque assay, but there are DENV strains that do not form plaques. For this reason, a PCR protocol able to detect and quantify DENV in the different kinds of samples employed for DENV study is of great value. In this study, a real-time PCR method suitable not only for clinical objectives but also for laboratory routine is described. Sequences from several strains of DENV comprising the four serotypes were aligned. A fragment located at the 5'UTR region of the virus genome was used to generate the primers and the probe for real-time PCR. This method was successfully used to identify and quantify distinct dengue virus strains and serotypes in clinical samples, in sera from patients infected with dengue virus, and in the mosquito Aedes aegypti, as well as to study virus replication in different cell lines.