Substrate phosphorylation by the kinases in Mycobacterium tuberculosis (MTB) is crucial for pathogenicity and the survival inside macrophages. Therefore, the identification of cognate kinase-substrate pairs is imperative. Our data through LC-MS/MS analysis showed that GarA (a known substrate for PknG) is phosphorylated at a unique site by two cytosolic kinases PknG and PknK, whereas both the kinases phosphorylate VirS (a known substrate for PknK) at two different sites. To establish biological significance of phosphorylation of GarA, we have demonstrated that recombinant M. smegmatis (MSMEG) over-expressing MTB specific kinase-substrate pairs and their mutants, alter the growth and intracellular survival of mycobacteria. These findings for the first time revealed that cytosolic serine/threonine kinases share GarA as a common substrate to persuade biological functions in mycobacteria.