Accurate determinations of acrosome reaction (AR) are fundamental to obtain physiologically relevant information of this essential event for sperm fertilization. For decades, the AR was exclusively studied in fixed preparation; more recently, new methods were developed to evaluate the AR in live sperm and in real time. These new technologies involved the use of genetically modified mice models, dyes with ion affinity, and fluorescent microscopy. In addition, these techniques allowed the reproduction field the possibility to follow the AR directly in the female tract under physiological conditions. Despite these advances, maintaining transgenic colonies is expensive for most research laboratories. Here, we present a methodology which can be mounted in a simple epifluorescence microscopy set in combination with appropriate filters enabling to follow the AR in real time. The implemented technique allows also to follow the AR in combination with [Ca2+]i, an ion directly involved in the regulation of this exocytotic reaction. In this chapter, we present in a simple way the methodology required to obtain AR/[Ca2+]i determinations using Fluo-4 and FM4-64 fluorescence recordings in live sperm under physiological and non-physiological stimuli. Also, we explain step by step the analysis and treatment to the images obtained during the recordings to determine AR quantitative information. The general methodology can be applied to measure other sperm parameters for which fluorescent dyes are available, such as intracellular pH (pHi) in combination with [Ca2+]i and/or the AR.