The evaluation of the intracellular calcium is essential in studying sperm capacitation and acrosome reaction, among other processes. Here, we describe the use of chlortetracycline (CTC) to study Ca2+ distribution in relation to the sperm capacitation state, and the evaluation of Ca2+ levels in spermatozoa using a low or a high-affinity Ca2+ dye (Rhod-5 N-AM and Fluo-4-AM, respectively) by flow cytometry. Rhod-5 N-AM staining method also allows the study of Ca2+ in subcellular compartments, whereas the changes in intracellular Ca2+ levels can be measured in spermatozoa loaded with Fluo-4-AM by plate reader fluorometry.