Amperometry is a commonly used technique for detecting the kinetics of single-vesicle exocytosis with excellent temporal and spatial resolution. However, different methods of analyzing the amperometric signals can produce conflicting conclusions. We developed an efficient automated method for kinetics analysis of single spikes that does not filter the data and therefore prevents distortion of the results. The algorithm assesses the signal-to-noise ratios (SNRs) and accordingly, separates the signals using an adjustable two-threshold calculation. This enables comparing data with different SNRs from different setups. The software also includes a complete statistical analysis, with an automated selection of the most appropriate statistical tests and a graphical representation. The algorithms can be used for any other experimental results requiring the separation of signals from noise, making this method useful for many applications.