Updated FTP location. Protein tyrosine phosphatases (PTPs) cooperate with protein tyrosine kinases to regulate intracellular tyrosine phosphorylation levels, and their dysfunction is associated with various diseases. However, due to the low abundance of PTPs in cells, comprehensive analysis requires an enrichment step prior to liquid chromatography/tandem mass spectrometry (LC/MS/MS). In this study, we designed and synthesized peptide probes for PTP pulldown assay using a non-hydrolyzable phosphotyrosine mimetic, 4-[difluoro(phosphono)methyl]-L-phenylalanine (F2Pmp). We found that different F2Pmp probes can enrich different PTPs, depending on the probe sequence. Furthermore, proteins containing a Src homology 2 (SH2) domain were enriched together. Importantly, probes containing phosphotyrosine instead of F2Pmp failed to enrich PTPs due to dephosphorylation during the pulldown step. This enrichment approach using peptides containing F2Pmp could be a generic tool for tyrosine phosphatome analysis without the use of antibodies.