Several analytical methods have been developed to study apoptosis (programmed cell death), many of which rely on fluorescence processes. 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 probe and method development 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 super-resolution microscopy, multiphoton excitation, and single cell-single molecule spectroscopy are quickly emerging. This updated article will explore several fluorescence approaches used in apoptosis studies as well as describe the mechanisms and hallmarks of the apoptosis cascade. Since apoptosis plays such an important role in both healthy and diseased organism function, the need to develop and apply sensitive analytical methods continues to be of the utmost importance.