Several analytical methods, many of which rely on fluorescence processes, have been developed to study apoptosis (programmed cell death). Apoptosis is a highly regulated biological event and is a vital process that helps regulate tissue growth, normal cell turnover, immune response, and tissue development. However, diseases such as cancer and heart disease are associated with malfunctions in the apoptosis machinery. There is therefore a need to elucidate the processes of apoptosis induction and inhibition. Fluorescence assays continue to play a major role in apoptosis assays, and developments in probe and fluorescent methods for assaying apoptosis are ongoing. There are several standard techniques, such as flow cytometry and confocal microscopy, for apoptosis study in cells. In addition, new techniques such as superresolution microscopy, multiphoton excitation, and single-cell−single-molecule spectroscopy are quickly emerging. This article explores several fluorescence approaches used in apoptosis studies as well as describes the mechanisms and hallmarks of the apoptotic cascade. As apoptosis plays a very important role in both healthy and diseased organism functions, the need to develop and apply sensitive analytical methods continues to be of the utmost importance.