A fast, sensitive and reliable flow cytometry-based (FACS = fluorescence activated cell sorting) immunofluorescence inhibition assay (FACS-IFI) for the detection of virus-specific antibodies in sera is described. The method was evaluated using sera from cattle experimentally infected with bovine viral diarrhea virus (BVDV). Virus-infected cells, which were fixed and permeabilized, were incubated with diluted sera from immunized or control animals. Monoclonal antibodies (mabs) against different viral proteins were added, and detected with ALEXA488-conjugated goat-antimouse antibodies. The fluorescence signals were detected by flow cytometry and determined as mean channel values. Results were expressed as percent fluorescence inhibition compared to standardized negative sera. The FACS-IFI test with sera from experimentally infected animals was highly sensitive and specific. Comparison of the FACS-IFI results with a commercially available blocking ELISA, an indirect ELISA and the standard serum neutralization test showed a strong correlation. Furthermore, the detection of protein-specific antibodies was possible using the FACS-IFI test.