Phosphatidylinositol 3,4,5 trisphosphate [PtdIns(3,4,5)P 3 ] is a potent membrane-bound signaling molecule transiently synthesized by phosphoinositide 3-kinase (PI3-kinase) in response to extracellular agonists. PtdIns(3,4,5)P 3 signals need to be strictly controlled. PtdIns(3,4,5)P 3 recruits and binds effectors that function in oncogenic signaling pathways. PtdIns(3,4,5)P 3 activates cell proliferation, growth, and migration as well as regulating insulin signaling. The inositol polyphosphate 5-phosphatase family of enzymes dephosphorylate and thereby modulate PtdIns(3,4,5)P 3 levels, attenuating PI3-kinase-dependent signaling. PtdIns(3,4,5)P 3 5-phosphatase enzyme activity can be assessed in vitro by analysis of the hydrolysis of radiolabeled or fluorescently labeled PtdIns(3,4,5)P 3 and in vivo by visualization of the recruitment and turnover of the PtdIns(3,4,5)P 3 -specific biosensor GFP-PH/ARNO or other PtdIns(3,4,5)P 3 binding proteins at the plasma membrane.