Tyrosine phosphorylation is a critical post-translational modification (PTMs) regulated by protein tyrosine phosphatases (PTPs). Dysregulation in PTPs activity is often associated with human diseases. Recently, much focus has been placed on PTPs in the context of liver metabolic diseases and liver cancer, as there is growing evidence suggesting that PTPs can regulate processes associated with the progression of metabolic dysfunction-associated steatotic liver disease (MASLD). PTP enzymatic activity is dependent on the cysteine residue, that resides within the conserved [I/V]HCSxGxGR[T/S]G motif, shared exclusively among all the PTPs. Cysteine is susceptible to oxidation by reactive oxygen species (ROS); thus, oxidation is an intrinsic mechanism that maintains PTP activity dynamics within a required range. Understanding the PTP activity across MASLD progression could shed light on the phosphorylation patterns, offering strategy that can reverse dysregulated kinase activities. Here, we perform sequential steps of chemical derivatization to lock physiological redox-regulated PTP states and exploit the conserved [I/V]HCSxGxGR[T/S]G motif to facilitate PTP purification. To achieve complete and specific PTP detection we utilized a targeted MS acquisition in a parallel reaction monitoring (PRM) mode for selective PTP identification, based on the upstream sequence of the [I/V]HCSxGxGR[T/S]G motif. The therapeutic success of PTMs inhibitors underscores potential of PTPs as targets for therapeutic interventions. Thus, the developed method is applied to human liver biopsies representing different stages of MASLD to uncover the PTPs repertoire, enabling dissection of intricate phosphorylation signaling pathways in the liver and guide clinical therapeutic interventions in MASLD.