Allele-specific enzyme inhibitors are powerful tools in chemical biology. However, few general approaches for the discovery of such inhibitors have been described. Herein is reported a method for the sensitization of protein tyrosine phosphatases (PTPs) to small-molecule inhibition. It is shown that mutation of an active-site isoleucine to alanine (I219A) sensitizes PTP1B to inhibition by a class of thiophene-based inhibitors. This sensitization strategy succeeds for both 'orthogonal' inhibitors, designed to be incompatible with wild-type PTP active sites, and previously optimized wild-type PTP inhibitors. The finding that the I219A mutation sensitizes phosphatase domains to a variety of compounds suggests that isoleucine 219 may act as a 'gatekeeper' residue that can be widely exploited for the chemical-genetic analysis of PTP function.