Protein tyrosine phosphatases (PTPs) play key roles in regulating tyrosine phosphorylation levels in cells. Since the discovery of PTP1B as a major drug target for diabetes and obesity, PTPs have emerged as a new and promising class of signaling targets for drug development in a variety of therapeutic areas. The routine use of generic substrate 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) in our hands led to the discovery of very similar and often not very selective molecules. Therefore, to increase the chances to discover novel chemical scaffolds, a side-by-side comparison between the DiFMUP assay and a chip-based mobility shift assay with a specific phosphopeptide was performed, on 1 PTP, using a focused set of compounds. Assay robustness and sensitivity were comparable for both the DiFMUP and mobility shift assays. The off-chip mobility shift assay required a longer development time because of identification, synthesis, and characterization of a specific peptide, and its cost per point was higher. However, although most potent scaffolds found with the DiFMUP assay were confirmed in the mobility shift format, the off-chip mobility shift assay led to the identification of previously unidentified chemical scaffolds with improved druglike properties.