Due to its slow growing nature, detecting Mycobacterium paratuberculosis (MAP) in biological samples can be problematic. Moreover, determining viability is fraught with complications and technical difficulties. Our aim was to employ real-time PCR as a means of resolving the fundamental challenges associated with determining viability and estimating mycobacterial numbers. Initially we developed a robust and efficient DNA extraction protocol for mycobacteria from milk and clinical samples. It was found that the combination of a guanidinium based lysis buffer, physical grinding and a silica based purification column worked optimally for mycobacterial DNA. Subsequently, a real-time PCR assay was designed and optimised for the IS900 region of MAP which had a sensitivity of 40cfu/ml (in less than 3 hours). Quantitative analysis was then carried out on artificially contaminated milk samples using this assay and the correlation between actual and predicted numbers of mycobacteria was assessed. Subsequently, a novel approach was used to assess mycobacterial viability using an assay based on real-time PCR and mycobacteriophages. After designing and optimising a real-time PCR for mycobacteriophage D29, the phage and mycobacterial host M. smegmatis were mixed in different ratios. In all cases the initial phage DNA levels were calculated by real-time PCR and samples were removed over time until all mycobacterial cells were lysed. DNA was extracted from samples removed at each time point and the total amount of phage DNA was determined using quantitative real-time PCR. As D29 can only infect living cells, a notable increase in phage DNA was used as an indirect measure of the viability of mycobacteria.We have shown that real time PCR is rapid, delivers reproducible quantitative information and offers a powerful alternative to conventional means of detecting mycobacteria in milk and clinical samples.