Inositol-1,4,5-trisphosphate (IP3) plays a central role in calcium signalling. Its production is catalyzed by phospholipase C enzymes, which can be activated through receptor stimulation. When investigating these signalling pathways, calcium measurement is not the most appropriate method to detect receptor activation, because several parallel processes can alter intracellular calcium concentrations. To solve this problem, we designed an IP3 probe, which is able to directly reflect the level of this compound, and thus allows a more accurate characterization of these signalling pathways. To construct this sensor, we used the human type-1 IP3 receptor, in which amino acids 224-605 are responsible for ligand binding. According to crystal-structure data, IP3 binding may lead to a conformational change of this protein domain. Based on this conformational change, we were aiming to construct a resonance energy transfer-based biosensor. In literature, similar probes can be found, but all of them have limitations. In particular, measurement of decreasing IP3 levels is uncertain, due to the high ligand affinity of these sensors. To eliminate this effect, we engineered two lower affinity mutants by impairing the binding site of the protein domain. The arginine in either position 265 or 269 was substituted to lysine (R265K and R269K, respectively). Finally, two appropriate luminescent chromophores were fused to the two ends of the protein domain to enable the monitoring of IP3 concentrations by fluorescence or bioluminescence resonance energy transfer. To compare our sensors, we needed an experimental system, in which an increase or decrease in IP3 concentration could be equally established. We created a modified HEK-293T cell line, which stably expressed a rapamycin-inducible phosphatidylinositol 4,5-bisphosphate (PIP2) depletion system. To achieve a stochiometric expression of its proteins, the components of this depletion system were cloned into a self-cleaving 2A-peptide containing vector. In addition, type-1 angiotensin receptor was transiently transfected to these cells, so IP3 concentration increased upon angiotensin II stimulus, while a decrease was seen after rapamycin treatment and consequent PIP2 degradation. Under these conditions both the wild type and the mutant sensors were able to show the increase in IP3 levels, but only the mutants were capable of detecting the decrease of IP3 concentration. Our data suggest that these sensors are suitable to investigate IP3 signalling more accurately by single cell imaging as well as in cell population measurements.