Most normal human cells have a finite replicative capacity and eventually undergo cellular senescence, whereby cells cease to proliferate. Cellular senescence is also induced by various stress signals, such as those generated by oncogenes, DNA damage, hyperproliferation, and an oxidative environment. Cellular senescence is well established as an intrinsic tumor suppressive mechanism. Recent progress concerning senescence research has revealed that cellular senescence occurs in vivo and that, unexpectedly, it has a very complex role in tissue repair, promoting tumor progression and aging via the secretion of various cytokines, growth factors, and enzymes. Therefore, the importance of biomarkers for cellular senescence has greatly increased. In 1995, we described the “senescence-associated β galactosidase” (SA-βgal) biomarker, which conveniently identifies individual senescent cells in vitro and in vivo. Here, we describe an updated protocol for the detection of cell senescence based on this widely used biomarker, which contributed to recent advances in senescence, aging and cancer research. We provide an example of detecting SA-βgal together with other senescence markers and a proliferation marker, EdU, in single cells.