The infection caused by Orthoflaviviruses is a global health concern and causes millions of infections and economical loss every year. Pathogenic Orthoflavivirus infecting human includes the Dengue virus (DENV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), West Nile virus (WNV) and Yellow fever virus (YFV) causing disease ranging from mild fever to severe manifestations including hemorrhagic fever and encephalitis. These Orthoflaviviruses causes similar symptoms and often co-infect and co-circulates in endemic areas which sometimes lead to misdiagnosis. Therefore, accurate and early diagnoses of Orthoflavivirus infections are inevitable. The current chapter describes the molecular and serological diagnostic techniques which are currently in use such as polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA), isothermal techniques like nucleic acid sequence-based amplification (NASBA), reverse transcription loop-mediated isothermal amplification (RT-LAMP) including others. These diagnostic methods were explored for their ease of use, minimum time involved in the assay, specificity, sensitivity, low cross-reactivity and false-positive results. The rapidity and sensitivity of techniques for differential diagnosis of Orthoflaviviruses is essential for proper disease management and thus improvements in the current techniques are needed for effective patient management.