Background: The purpose of this study was the development of multiplex fluorescence microsphere immunoassay (FMIA) for the detection of Rift Valley fever virus (RVFV) IgG and IgM antibodies by incorporation of antigens from whole cell lysates and purified virions from MP12. Methods and Findings: Viral antigens were derived from whole cell material prepared from BHK cells infected with MP-12 (C-MP12). Virions from cell-free culture supernatant were purified on a sucrose cushion (V-MP12). The antigen material was treated with Triton X-100 or NP-40 prior to coating onto Luminex beads. V-MP12 and C-MP12 were assembled into a multiplex and tested in sheep and cattle infected with wild-type RVFV and the vaccine strain MP12. In these studies, V-MP12, C-MP12, and recombinant antigens (Gn, NSs, NSm, N) conjugated beads used in multiplex FMIA and the maximum level of IgG response to sheep in V-MP12 was recorded. IgG reactivity for the antigen targets varied with V-MP12>C-MP12. The positivity-negativity were evaluated by calculating S/P ratio using serum from RVFV-negative sheep (n=100) and negative cattle (n=100). The reaction of monoclonal anti-RVFV N antibody has been tested against N, V-MP12, and CMP12 by FMIA and all the antigens were reactive to antibody N>V-MP12>C-MP12. Conclusions: The experimental results demonstrate that the incorporation of V-MP12 and/or C-MP12 in the FMIA provides an improved sensitivity and specificity of FMIA with internal confirmation (i.e. multiple antigen targets) thus providing advantages over the traditional ELISA.