Phospholipids such as phosphatidylinositol(4,5)bisphosphate (PI(4,5)P₂) influence the activity of many ion channels. PI(4,5)P₂ affinity may vary considerably between channels as deduced from application of the soluble PI(4,5)P₂ analog di8-PI(4,5)P2. However, it is a matter of debate whether di-8PI(4,5)P₂ functionally mimics endogenous PI(4,5)P₂. Because PI(4,5)P₂ affinity may be the most important factor determining the impact of PI(4,5)P₂ signalling on proteins, and ion channels in particular, we were interested in quantifying PI(4,5)P₂-sensitivity in the intact cellular environment. We have shown that the voltage-sensor containing phosphatase Ci-VSP, which converts PI(4,5)P₂ into PI(4)P when activated by depolarization, allowed the reversible and gradual titration of endogenous PI(4,5)P₂ in intact cells. Kir currents, monitored by whole cell patch-clamp in CHO cells co-expressing Ci-VSP, declined upon depolarization due to PI(4,5)P₂ depletion by Ci-VSP activation and recovered upon repolarization as a result of endogenous PI(4,5)P₂ resynthesis. Steady state channel deactivation upon gradual Ci-VSP activation, as a readout for the channel-PI(4,5)P₂ affinity, indicated high affinity for Kir2.1, intermediate affinity for Kir1.1 and low affinity for Kir3 channels, which is in well agreement with previous studies. Furthermore, reduced or dynamically strengthened PI(4,5)P₂-affinity by mutation (Kir2.1R228Q) or GPCR activation (G_bg-effect on Kir3) were detected by shifted steady state channel deactivation curves towards hyperpolarization or depolarization, respectively. Finally, quantifying the controversial PI(4,5)P₂ affinities of two fluorescent PI(4,5)P₂-binding domains, PLCd1-PH and tubby-CT, with Ci-VSP, revealed that tubby-CT exhibits a lower affinity than PLCd1-PH, indicating suitability to detect small PI(4,5)P₂ changes reliable. These results establish Ci-VSP as a powerful molecular tool for quantifying PI(4,5)P₂ sensitivity in the living cell. Supported by Deutsche Forschungsgemeinschaft (DFG Ol 240/2, SFB593 TPA12 to D.O.)