The implementation of intracytoplasmic sperm injection (ICSI) in the field of assisted reproduction has allowed fertilization in couples with previous fertilization failure. However, complete fertilization failure after ICSI shows a prevalence of up to 3%. Moreover, low or moderate fertilization (<30%) can be observed for some patients. In such cycles of bad prognosis, the underlying problem is most likely related to a failure of oocyte activation. In vivo, a sperm-derived enzyme mediates the physiological stimulus of the complex process of oocyte activation. In vitro, a complete fertilization failure can eventually be compensated by several artificial oocyte activation (AOA) techniques, all of which aim for an increase in internal Ca²⁺. AOA is only applied with the proper indication and not as a routine method. The lack of standardized AOA methodologies led to the development and launch of a ready-to-use AOA solution and the number of publications dealing with AOA is constantly increasing. This chapter describes a proven protocol for AOA that has been used in clinical practice for more than two decades.