The extraction of proteins from Mycobacterium avium subsp. paratuberculosis (M. ptb) or Mycobacterium avium subsp. avium (M. av) bacterial cells, using Triton X-114 (TX-114) phase-separation, has been used to identify proteins associated with the mycobacterial envelope. In this study we demonstrate that the method was suitable for selective extraction of proteins from the surface of bacterial cells and then further partition them into either one or both detergent phases. Extracted proteins were separated by gel electrophoresis and identified by mass spectrometry and bioinformatic interrogation. The detergent phases comprised mostly proteins predicted to be non-cytoplasmic, whilst the vast majority of proteins in the aqueous phases were predicted to be located in the bacterial cytoplasm. Using this approach we have identified 70 proteins from TX-114 extracts of both M. ptb and M. av. Several proteins were identified that appeared to be unique to M. ptb extracts. Three proteins identified in pathogenic M. ptb (AhpC, Tpx and BcpB) were classified as being detoxification proteins, possibly suggesting that these proteins may form part of this organism’s defence against oxidative attack and as such may represent potential targets for anti-mycobacterial drug development. The strong response of sera from goats with Johne’s disease (JD) to detergent-rich phase proteins of both M. ptb and M. av are consistent with previous observations where strong immune responses were associated predominantly to envelope associated proteins. The separation of TX-114 extracts of M. ptb and M. av underlines the importance of this method in pre-fractionating the bacterial proteome.