Background and Aim:Brucellosis, paratuberculosis (PTb), and infections caused by small ruminant lentivirus (SRLV), formerly known as caprine arthritis encephalitis virus (CAEV), adversely affect goat production systems. Nonetheless, commonly used diagnostic tests can only determine one analyte at a time, increasing disease surveillance costs, and limiting their routine use. This study aimed to design and validate a multiplex assay for antibody detection against these three diseases simultaneously.Materials and Methods:Two recombinant proteins from the SRLV (p16 and gp38), the native hapten ofBrucella melitensis, and the paratuberculosis-protoplasmic antigen 3 fromMycobacterium aviumsubsp. paratuberculosis (MAP) were used to devise and assess a multiplex assay. Conditions for the Luminex®multiplex test were established and validated by sensitivity, specificity, repeatability, and reproducibility parameters. Cut-off points for each antigen were also established.Results:The 3-plex assay had high sensitivity (84%) and specificity (95%). The maximum coefficients of variation were 23.8% and 20.5% for negative and positive control samples, respectively. The p16 and gp38 SRLV antigens are 97% and 95%, similar to the CAEV sequence found in GenBank, respectively.Conclusion:The multiplex test can be effectively used for the simultaneous detection of antibodies against SRLV, MAP andB. melitensisin goats.