Arthropod-borne viruses (arboviruses) belonging to the Flavivirus genus of the Flaviviridae family, are a major public health threat in tropical and subtropical regions, and have recently become a medical concern in temperate zones. Most flaviviruses are classified as zoonotic viruses. Human flavivirus infections can be asymptomatic, responsible for unspecific symptoms in the first few days following infection, or responsible for severe complications potentially resulting in death. During the first days following symptom onset, laboratory diagnosis of acute human flavivirus infection is mainly based on molecular detection of the viral genome by RT-PCR methods, followed by the capture of specific antibodies using serological tests after the first week of infection. The detection of antibodies that have virus neutralizing activity can be used to confirm flavivirus infection. However, human flavivirus infections induce the production of cross-reactive antibodies, often making serology inconclusive. Indeed, serological diagnosis of flavivirus infection can be hampered by a patient’s history of flavivirus exposure, particularly in regions where multiple antigenically related flaviviruses co-circulate. We focus our mini review on conventional immunoassays that allow the diagnosis of major flavivirus-associated human infections in basic, routine and high-profile central health centers; and the interpretation of diagnostic serology tests for patients living within different epidemiological situations.