Although the phosphorylation of serine (S), threonine (T), and tyrosine (Y) is well-established, arginine phosphorylation (pR) has recently garnered significant attention due to its crucial role in bacteria pathogenicity and stress response. Mycolicibacterium smegmatis, a nonpathogenic surrogate of Mycobacterium tuberculosis, serves as a model for studying mycobacterial pathogenesis. A recent proteomics study identified six pR proteins in M. smegmatis. To gain a more comprehensive understanding, we performed pR profiling using mass spectrometry in combination with two distinct phosphopeptide enrichment strategies: titanium-immobilized metal ion affinity chromatography (Ti