Protein tyrosine phosphatases (PTPs) have a central role in signal transduction. Together with protein tyrosine kinases (PTKs), PTPs oversee a wide array of fundamental biological processes. PTP dysfunction is connected with prolonged phosphorylation signaling, which can in turn contribute to a plethora of human diseases, including metabolic diseases and cancers. The phosphatase activity relies on the catalytic cysteine located within the conserved and exclusive HCX5R motif, present in all PTPs. Cysteine is susceptible to oxidation by reactive oxygen species (ROS), and the PTPs are thereby switched on and off, transiently, and reversibly. While a large number of studies have converged on dysregulated phosphorylation signaling as disease cause, few publications focus on phosphatase activity despite only 37 such phosphatases being encoded in the human genome. Understanding the tissue-specific and disease-relevant PTP activity could also shed light on the systems level phosphatase regulation, which can simultaneously correct for multiple dysregulated kinase activities within the same organ. Here we exploit the conserved HCX5R motif surrounding the catalytic cysteine to design a PRM panel of 47 catalytic peptides found on all 37 human PTPs. In combination with antibody-mediated catalytic site peptide retrieval, this novel targeted MS strategy could be applied to cultured cells and tissue specimens alike, to enable discovery and quantification of all human PTPs, collectively known as the PTPome profile, starting from needle biopsy material. We envision such data will not only guide the clinical therapeutic interventions surrounding phosphorylation signaling but may also be critical to dissect intricate signaling pathways in health and disease.