Tuberculosis (TB) is a highly infectious disease, that was responsible for ~ 1.3 million deaths in 2012. The causative agent of this disease is the pathogenic bacterium Mycobacterium tuberculosis (Mtb). With almost 9 million new cases of TB diagnosed every year this disease remains a major global threat to human health. Indeed the current threat to human health is further exacerbated by the recent emergence of several drug resistant strains of Mtb, including a strain that is resistant to all currently available antibiotics. Hence, there is an urgent need for the discovery and development of novel drugs against this pathogen. Recently, several novel antibiotics have been identified, which exhibit bactericidal activity against multi-drug resistant isolates of Mtb. Significantly, these compounds target the unfoldase component (ClpC), which is the molecular motor of the ClpCP proteolytic machine, thereby validating this machine as a novel drug target. Currently however, little is known about the physiological role of the ClpCP protease in Mycobacteria. Moreover the precise mode-of-action of these compounds is only poorly defined, although they are believed to dysregulate ClpC function through interaction with the N-terminal domain (NTD) of ClpC. Interestingly, the NTD of many AAA+ proteins is a docking site for adaptor proteins. Here we describe the characterisation of a putative ClpC adaptor protein (ClpS) from Mycobacterium smegmatis (Msm) that may play a role in the N-end rule pathway in this bacterium. Preliminary studies show that MsmClpS not only binds model N-end rule substrates, but also modulates the activity of the ClpC unfoldase. In addition, we have isolated and identified a number of potential N-end rule substrates from Msm cells. These data will help define the physiological role of the ClpCP protease and the N-end rule pathway in mycobacteria.