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Purpose: West Nile virus (WNV) and dengue virus (DENV) are emerging arthropod-borne flaviviruses that represent an immense global health problem. Therefore, there is need for improved diagnosis of these infections for timely patient management, to prevent the spread of flaviviral infections as well as to understand the virus and antibody dynamics in humans as well as animal models. In this study, Luminex-based WNV E-protein microsphere immunoassay (MIA) was developed and optimized for detection of anti-WNV IgG and IgM antibodies in mice using assay parameters such as serum heat-inactivation (HI) and-dilution. In addition, an in-house newly developed Luminex-based assay for detection of anti-DENV IgG and IgM antibodies was further validated using different serum panels and was further optimized for improved sensitivity and specificity which is critical for accurate diagnosis of DENV infection. Similarly, inhouse newly developed Polymerase Chain Reaction-Microsphere Bead Assay (PCR-MBA) was further validated for detection of DENV serotypes and WNV. Methods: Magnetic carboxylated microspheres were coupled to purified rWNV-E protein and WNV E-MIA was conducted using serial dilutions of HI and non-HI (NHI) serum samples collected at days 0, 3, 6, 8, 10 and 24 from mice inoculated with WNV. In-house newly developed DENV MIA was validated using different serum panels from Hawaii, Vietnam and Niue as compared to gold standard PRNT assay and in-house DENV IgM Capture ELISA (MAC-ELISA) and a U.S. FDA approved InBios DENV IgM Capture ELISA. To improve the specificity and sensitivity of DENV MIA, the effect of assay parameters such as serum dilution, the use of alternative blocking agents such as PVA, PVP and 5-10% animal serum in serum diluents as well as assay buffers and the type and concentration of secondary antibody on the non-specific binding was studied. An in-house newly developed PCR-MBA was further validated for differential detection of DENV serotypes using CDC DENV panel samples as compared to gold standard CDC DENV 1-4 real-time RT-PCR assay. For validation of PCR-MBA for detection of WNV, WNV cDNA was diluted serially (10-fold dilutions) from 3 × 107 PFU/ml to 0.3 PFU/ml and WNV specific qRT-PCR and PCR-MBA was conducted. Data was analyzed to assess the sensitivity and specificity of PCRMBA for detection of WNV as well as for differential detection of DENV serotypes. Results: In WNV E-MIA, serum HI significantly enhanced detection of IgM and IgG antibodies as compared to NHI serum. Anti-WNV IgM and IgG antibodies in HI sera were detected earlier at day 3 and IgM antibodies persisted up to day 24 after infection. HI serum at 1:20 dilution was found to be optimal for detection of both IgM and IgG antibodies as compared to higher serum dilutions. Further, addition of exogenous complement to the HI serum decreased the WNV E-MIA sensitivity. Results of validation of DENV MIA demonstrated that DENV MIA is 100% and 71-100% sensitive for detection of anti-DENV IgG and IgM antibodies, respectively from human serum samples. However, the specificity of DENV MIA was found to be 33-100% and 11-98% for detection of anti-DENV IgG and IgM antibodies respectively. The low specificity of DENV MIA for samples from Vietnam was not due to syphilis antibodies present in these serum samples. Use of alternative blocking agents such as PVA and PVP, 5-10% animal serum in serum diluents did not increase the specificity of DENV IgM MIA. Higher serum dilution as well as pretreatment of serum samples with high concentration of BSA or BSA-coated beads reduced the assay interference to some extent in DENV IgM MIA. The use of monoclonal IgM secondary antibody for detection of anti-DENV IgM antibodies resulted in low BSA IgM MFI for IgM false-positive samples as compared to polyclonal IgM secondary antibody in DENV MIA. However, the IgM MFI for true IgM positive samples was reduced using monoclonal antibody as compared to polyclonal secondary antibody. The specificity of PCR-MBA for detection of four DENV serotypes as compared to 'gold standard' CDC DENV 1-4 real time RT-PCR assay was found to be 100%, whereas the sensitivity of PCR-MBA was found to be varied from 50-100% for DENV-1, 29-100% for DENV-2, 100% for DENV-3 and 80-100% for DENV-4 using CDC DENV panel samples. PCR-MBA for detection of WNV is specific and more sensitive as compared to WNV-specific qRT-PCR. Moreover, low background MFI was observed for the other arboviruses included in PCR-MBA and thus indicated negligible probe cross reactivity for the DENV serotypes and WNV. Conclusions: Serum-HI and optimal dilution enhanced WNV E-MIA sensitivity by eliminating the complement interference. This optimized WNV E-MIA can be used for detecting low-titer anti-WNV antibodies during early and late phase of infection in mice. Validation of DENV MIA using different serum panels demonstrated that sensitivity and specificity of immunoassays may differ according to the origin of samples. Minimizing the serum concentration as well as the absorption of sera with high concentration of BSA or BSA coated beads decreased the non-specific binding in DENV IgM MIA. Moreover, the results of this study conclude that sample pretreatment or the use of alternative blocking agents will vary for immunoassays. Therefore, it is important to optimize the blocking conditions for newly developed immunoassays to avoid the falsepositive results. PCR-MBA efficiently detected and differentiated the DENV serotypes from CDC DENV panel samples as well as WNV. PCR-MBA can be further optimized for improved sensitivity for detection of dengue serotypes and WNV by changing the concentration of probe coupled, concentration of the input RNA, hybridization temperature and time.