In recent years it became clear that phosphoinositides (PI) are not only structural lipids in membranes, but they also have important roles in several cellular functions ranging from mediating signalling cascades in the cell by binding to effector proteins, through regulation of ion channels to cell movements. Reversible phosphorylation of the inositol ring at positions 3, 4 and 5 results in the synthesis of seven different PIs. Their levels in the plasma membrane, endomembranes and in the cytoplasm can influence these processes, thus measuring the level of these lipids in living cells can help us to better understand their distinct functions. Our aim was to develop a highly sensitive method which enables us to follow the dynamic change of these lipids in living cells. For this we performed bioluminescence resonance energy transfer (BRET) measurements between various luciferase-labeled PI-binding domains and a plasma membrane-targeted Venus (yellow fluorescent protein) in HEK-293T cells. To monitor the inositol lipid pools the following domains were used: the PH domain of PLCδ1 for PIP2, the 2xPH domain of OSH2 for PI4P and the PH domain of BTK for PIP3. To measure cytoplasmic IP3 level a recently developed intramolecular BRET sensor, based on the ligand binding domain of the type-1 IP3 receptor, was used. As expected, stimulation of the cells expressing type-1 angiotensin receptor with 100 nM angiotensin II resulted in a transient response including the decrease of the plasma membrane PI4P and PIP2 level, and increase of the cytoplasmic IP3 concentration, but no change of the PIP3 level. In contrast, more robust, sustained responses could be recorded if 100µM carbachol was applied to stimulate cells expressing M3 muscarinic acethylcholine receptors indicating that cells are able to synthetize huge amount of PI4P and PIP2 upon agonist stimulation. Stimulation of the cells expressing EGF receptor with 100 ng/ml EGF resulted in a sustained increase of the PIP3 and a slowly developing IP3 signal. Both PI4P and PIP2 levels were decreased, but in contrast with Gq-activating hormones, decrease of the PI4P pool was continuous and significantly higher compared to the transient decrease of the PIP2 level. Our results indicate that this approach is highly sensitive and capable of quantitative and dynamic characterization of inositol lipid changes upon stimulation of the cells with various compounds. Selective measurements of the various lipid pools may result in the discovery of differences between stimuli and therefore the role of inositol lipid pools and inositol modifying enzymes during the activation process.