The integrated total charge (Q) of individual redox current spikes (Figure , Table ) was used for calculating the average charge density (Q/LUV size). The linear relationship when plotting the charge density against the ACh concentration encapsulated within the LUVs establishes a calibration curve for cellular recordings (Figure ). The absolute quantification of ACh released at exocytosis was then performed for each spike type using the calibration curve. Figure , illustrates that the analysis indicates both complex plateau-shaped and full sharp events release an average of 8400 ± 320 ACh molecules (mean ± SEM), implying full exocytosis and complete release of vesicular ACh content. This estimation align with the consensus of 5,000 to 10,000 ACh molecules per vesicle. In contrast, the most prevalent, 'sharp spikes', release an average of 4700 ± 160 ACh molecules (mean ± SEM). This indicates partial exocytosis release, consistent with earlier glutamate measurements for this spike type. Figure shows that pooling all release events detected into a histogram, a LogNormal equation fit estimates an average release of 5800 ACh molecules across all spike modes. Assuming an internal vesicle solution volume of 20 zL, we estimate the original vesicular ACh concentration to be 0.7 ± 0.03 M, which is similar to findings for storage of glutamate and catecholamine in secretory vesicles. In summary, we present the development of an ACh biosensor and its application for monitoring the temporal dynamics of quantal release during exocytosis from presynaptic sites at human differentiated cholinergic cells with sub-millisecond precision. The high temporal resolution of the sensor facilitated the study of drug effects on exocytosis activity and quantal release, as well as the identification of various dynamic exocytosis modes. This ultrafast technology offers a novel approach to studying the regulatory aspects of fusion pore-controlled release and provides insight into the ACh system involved in both healthy neurotransmission and neurological disorders.