Our structure-based drug discovery program within the field of protein-tyrosine phosphatases (PTPs) demands delivery of significant amounts of protein with extraordinary purity specifications over prolonged time periods. Hence, replacement of classical, multi-step, low-yield protein purifications with efficient affinity techniques would be desirable. For this purpose, the highly selective PTP1B inhibitor 2-(oxalyl-amino)-4,5,6,7-tetrahydro-thieno[2,3-c]pyridine-3-carboxylic acid (OTP) was coupled to epoxy-activated Sepharose 6B (OTP Sepharose) and used for one-step affinity purification of tag-free PTP1B. The elution was performed with a combined pH and salt gradient. Importantly, since OTP Sepharose binds PTP1B with an intact active site only, the method ensures that the purified enzyme is fully active, a feature that might be particularly important in PTP research.