Cellular senescence is a physiological phenomenon by which normal cells irreversibly lose their proliferative potential. Understanding the mechanisms of senescence may be helpful to elucidate the causes of ageing and for the development of anti-tumor drugs. As an indicator of senescent cells, the senescence-associated beta-galactosidase (SA-beta-Gal) activity at pH 6.0 has been used widely for fundamental senescence research. However, the conventional SA-beta-Gal assay is not optimal for evaluating complex three-dimensional multicellular samples. We established a highly sensitive method for the direct quantification of the SA-beta-Gal activity of individual multicellular spheroids within 6 min, based on scanning electrochemical microscopy (SECM). Using SECM, we successfully detected the differences in SA-beta-Gal activity between MCF-7 spheroids undergoing all-trans retinoic acid (ATRA)-induced senescence and control spheroids. The SECM-based method demonstrated in this study may be widely applicable to characterize senescence processes involved in physiology and pathology. (C) 2015 Elsevier Ltd. All rights reserved.