Cellular communication is a critical process that relies on exocytosis, during which cells release stored chemical messengers contained within intracellular nanoscale vesicles (50-500 nm in diameter). Before this occurs, the vesicle membrane must open and form a fusion pore, allowing its contents to be released into the extracellular space. This subcellular process involves various biomolecules, such as lipids and proteins, within the membrane, and any changes in their levels can impact dynamic pore formation and, consequently, the exocytosis process. Due to their small size, intracellular location, and sensitivity, direct studies of vesicles are challenging yet highly valuable. One of these crucial biomolecules is phosphatidylinositol-4,5-bisphosphate (PIP2), a lipid involved in membrane dynamics and related processes including exocytosis. In this study, we employed a combination of sensitive confocal microscopy and vesicle impact electrochemical cytometry (VIEC)-a novel amperometric technique using microelectrodes (D, 33 μm)-to test the hypothesis that elevated PIP2 levels regulate vesicle membrane properties and indirectly influence the exocytosis process. To investigate this, we used nanoscale vesicles isolated from neural cells as a biological model system. First, imaging analysis revealed that high PIP2 levels led to its accumulation in both cell and vesicle membranes, where it also participates in exocytosis. Next, direct analysis of PIP2-treated and untreated single nanoscale vesicles using VIEC demonstrated that while the vesicle content (i.e., the number of stored catecholamines) remained unchanged after PIP2 treatment, the vesicle opening dynamics were altered compared to the control. Specifically, our results showed that the vesicle opening rate increased by 1 ms, and the duration of vesicle opening extended from 5.7 to 6.9 ms in PIP2-treated vesicles compared to the control. In addition to the recognized roles of PIP2, these findings indicate that an extra level of PIP2 modulates the vesicle opening rate and suggest that PIP2 enhances membrane stability while delaying the vesicle opening process. Interestingly, this observation aligns with previous experimental and computational studies, which reported that abnormally high levels of PIP2 or other lipids can modify membrane properties and then exocytosis too. In our study, we observed this effect for PIP2 at abnormal levels through single vesicle electroanalysis. Furthermore, these results open a new way of investigating similar membrane components and their roles in disease mechanisms and cellular processes.