<p dir="ltr">Protein phosphorylation is a ubiquitous post-translational modification controlled by the opposing activities of protein kinases and phosphatases, which regulate diverse biological processes in all kingdoms of life. One of the key challenges to a complete understanding of phosphoregulatory networks is the unambiguous identification of kinase and phosphatase substrates. Liquid chromatography-coupled mass spectrometry (LC-MS/MS) and associated phosphoproteomic tools enable global surveys of phosphoproteome changes in response to signaling events or perturbation of phosphoregulatory network components. Despite the power of LC-MS/MS, it is still challenging to directly link kinases and phosphatases to specific substrate phosphorylation sites in many experiments. Here we described two methods for the LC-MS/MS-based characterization of protein phosphatases and kinases. The first is an <i>in-vitro</i> method designed to probe the inherent substrate specificity of kinase or phosphatases. This method utilizes an enzyme reaction with synthetic peptides, serving served as substrate proxies, coupled with LC-MS/MS for rapid, accurate high-throughput quantification of the specificity constant (<i>k</i>_<em>cat</em><i>/K</i>_<em>M</em>) for each substrate in the reaction and amino acid preference in the enzyme active site, providing insight into their cellular roles. The second couple’s auxin-inducible degradation system (AID) with phosphoproteomics for protein functional characterization. AID is a surrogate for specific chemical inhibition, which minimizes non-specific effects associated with long-term target perturbation. Using this system, we demonstrate-PP2A in complex with its B-subunit Rox Three Suppressor 1 (PP2A^Rts1) contributes to the phosphoregulation of a conserved fungal-specific membrane protein complex called the eisosome. By maintaining eisosomes in their hypophosphorylated state, PP2A^Rts1 aids fungal cells in preserving metabolic homeostasis. This work demonstrates the power of mass spectrometry as a critical tool for protein functional characterization.</p>