PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a phosphatidylinositol triphosphate 3-phosphatase that counteracts phosphoinositide 3-kinases and has subsequently been implied as a valuable drug target for diabetes and cancer. Recently, we demonstrated that VO-OHpic is an extremely potent inhibitor of PTEN with nanomolar affinity in vitro and in vivo. Given the importance of this inhibitor for future drug design and development, its mode of action needed to be elucidated. It was discovered that inhibition of recombinant PTEN by VO-OHpic is fully reversible. Both K _m and V _max are affected by VO-OHpic, demonstrating a noncompetitive inhibition of PTEN. The inhibition constants K _ic and K _iu were determined to be 27 ± 6 and 45 ± 11 nM, respectively. Using the artificial phosphatase substrate 3-O-methylfluorescein phosphate (OMFP) or the physiological substrate phosphatidylinositol 3,4,5-triphosphate (PIP₃) comparable parameters were obtained suggesting that OMFP is a suitable substrate for PTEN inhibition studies and PTEN drug screening.