Protein tyrosine phosphatases (PTPs) are key regulators in living systems and thus are attractive drug targets. The development of potent, selective PTP inhibitors has been a difficult challenge mainly due to the high homology of the phosphotyrosine substrate pockets. Here, a strategy of dynamic substrate enhancement is described targeting the secondary binding sites of PTPs. By screening four different PTPs from bacterial (MptpA) and human origin (PTP1B, HePtp, Shp2) with this assay, specific fragments were identified. One highly specific fragment that binds to the secondary site of Mycobacterium tuberculosis protein tyrosine phosphatase A (MptpA) was characterized in order to validate the assay concept. Finally by covalently linking the secondary fragment to a phosphotyrosine mimetic, a moderately active but highly specific inhibitor of MptpA was obtained.