This study aimed to develop a multiplex quantitative PCR (qPCR) assay for the detection of Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of paratuberculosis disease, a chronic and endemic infectious disease affecting ruminant livestock worldwide. Infected animals may remain asymptomatic for years while intermittently shedding MAP into their environment through feces, contributing to ongoing transmission. To develop a robust multiplex qPCR assay, we reviewed all TaqMan qPCR studies published since 1990 and selected 18 primer-probe combinations targeting the MAP-specific F57 gene and the repetitive sequence elements ISMAP02 and IS900. In samples with moderate to high MAP levels, all combinations performed well, with only minor differences in analytical performance. However, in low-abundance samples, several TaqMan designs yielded unreliable results, indicating limited specificity in complex matrices. Among the evaluated assays, the IS900-Herthnek design demonstrated significantly higher diagnostic sensitivity, detecting MAP in 81% of low-abundance samples, compared to 0% and 3% for the IS900-Kim and IS900-Slana assays, respectively. For ISMAP02, only the ISMAP02-Sevilla assay produced reliable results. For F57, Herthnek provided the most consistent and accurate quantification. Similar trends were observed in environmental sample testing. Based on these findings, we recommend a multiplex qPCR assay incorporating the IS900-Herthnek, ISMAP02-Sevilla, and F57-Herthnek TaqMan designs for the detection of MAP in fecal and environmental samples. This combination offers high analytical sensitivity and specificity, making it a valuable and accurate tool for the diagnosis of paratuberculosis and for environmental surveillance on dairy farms to identify herds potentially harboring MAP-infected animals.IMPORTANCEMycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease (JD) in ruminant livestock industries and has been associated with Crohn's disease in humans. Emerging scientific evidence also links MAP to other human conditions, including inflammatory bowel disease, autoimmune disorders, colorectal cancer, and Alzheimer's disease. This potential public health threat has intensified interest in developing more sensitive diagnostic tools and effective control strategies to eradicate MAP from dairy herds. Infected ruminants typically remain in the subclinical stage of the disease for 2-5 years, during which they shed MAP in their feces and contaminate the environment. Diagnosis during this stage is particularly challenging, as the pathogen evades the host's immune response, rendering serological tests insufficiently sensitive. In contrast, fecal PCR offers greater sensitivity than serum ELISA and traditional culture methods. Multiplex quantitative PCR is especially promising due to its high specificity and sensitivity for detecting MAP-infected animals and identifying herds with active shedders. Herd-level environmental screening, followed by individual animal testing, represents a robust national biosecurity strategy. This approach is a critical step toward reducing MAP transmission and improving herd health within the dairy industry.