Brucellosis is a zoonotic disease that is caused by the species of bacteria, Brucella affecting goats and other livestock, that leads to economic deprivation and the likelihood of human transmission. This study sought to develop a highly sensitive and specific TaqMan® real-time PCR assay integrating hydrolysis probes for multiplex assay to differentiate active Brucella infection from passive shedding in goats targeting specific genes associated with Brucella melitensis. Using discontinuous conserved sequences, primers were designed for B. melitensis genes i.e. omp25, omp31, and IS711 with their compatible fluorescent dyes (Hex, FAM, Texas Red), and quencher molecules (BHQ1, BHQ1, BHQ2) respectively enabling multi-target detection within the same assay. The sensitivity of the multiplex assay for determining the Limit of Detection was evaluated by log 10 serial dilutions of Brucella nucleic acid (DNA, showing the presence of bacteria) and (RNA showing the presence of live bacteria), for each gene and optimized by probe, primer, and template titrations. LOD calculated for DNA was 8.53×10 10 (dil.1) to 8.53×10 7(dil.3) for omp25-HEX, = 1.32×10 11 (dil.1) to 1.32×10 8 (dil.3) for omp31-FAM, 1.08×10 11 (dil.1) to 1.08×10 7 (dil.4) for IS711-Texas Red. The lowest detectable limits were 8.53×10 7 , 1.32×10 8 , and 1.32×10 8 copies, respectively. For RNA (cDNA) LOD calculated was 1.58×10 10 (dil.1) to 1.58×10 7 (dil.3) for omp25-HEX, 2.45×10 10 (dil.1) to 1.8×10 6 (dil.4) for omp31-HEX, 2.0×10 10 (dil.1) to 2.0×10 6 (dil.4) for IS711-Texas Red. The lowest detectable limits were 1.58×10 7, 1.8×10 6 and 2.0×10 6 copies, respectively.LOD calculated from the cloned plasmid (target genes were cloned into pGEM-T Easy vector) was 2.6×10 4, 1.8×10 5 , 1.6×10 4 in simplex qPCR assay and 2.6×10 4, 1.8×10 5 , 1.6×10 5 in multiplex QPCR assay for omp31, omp21, IS711 genes respectively, making it easier to distinguish between animals that are actively shedding and those that are passively releasing non-viable Brucella. The specificity of the assay was confirmed through validation by comparing the nucleic acids from Brucella melitensis culture with Escherichia coli (Gram-negative) and Staphylococcus aureus, (Gram-positive) with the latter generating no specific response. This assay has the potential to supplement epidemiological research to ascertain the true prevalence of Brucella infection in goats. It also shows promise as a replacement for culture-based tests.