Polyacrylamide gel electrophoresis (PAGE) is a versatile technique widely used in molecular biology for the separation of biomolecules based on size and charge. While it is traditionally applied to proteins and nucleic acids, recently, PAGE has been adapted for the analysis of inositol polyphosphates and their pyrophosphate derivatives, which play multiple roles in cellular signaling. Studying these molecules presents analytical challenges due to their small size, high charge density, and low concentrations in biological samples. In this work, we describe an optimized PAGE protocol for the separation and detection of inositol polyphosphates and inositol pyrophosphate. The method uses high-concentration polyacrylamide gels and toluidine blue staining to achieve sensitive detection at nanomolar levels. The simplicity and cost-efficiency of the approach make it accessible to most laboratories. This method provides a reliable tool for investigating inositol-phosphate-based signaling pathways and their involvement in cellular processes.