Mycobacterial STPKs are responsible for orchestrating phosphorylation-dependent signaling cascades that mediate bacterial growth and environmental adaptation. Recent advances in MS-based phosphoproteomics have significantly expanded the candidate substrate lists for individual mycobacterial STPKs. Integration of the available phosphoproteomic datasets provide new insights into the functional roles of specific STPKs in cell physiology. Future research should focus on in vivo phosphorylation network reconstruction to expose the fundamental signaling pathways in mycobacteria. Linking STPKs with their physiological substrates may reveal novel antimycobacterial agents.