Senescence-associated β-galactosidase (SA-β-gal) is a biomarker for senescent cells, detected at pH 6.0. This study shows that SA-β-gal activity is derived from lysosomal β-galactosidase encoded by the GLB1 gene. In patients with GM1-gangliosidosis, who lack functional lysosomal β-galactosidase, SA-β-gal activity is absent despite replicative senescence. Similarly, reducing GLB1 mRNA levels using RNA interference in normal fibroblasts or HeLa cells led to senescence without SA-β-gal activity. These findings indicate that SA-β-gal activity is not required for senescence but is instead a result of increased lysosomal β-galactosidase levels in senescent cells. The study confirms that lysosomal β-galactosidase is the source of SA-β-gal activity, and that its increased expression in senescent cells contributes to the detection of SA-β-gal at pH 6.0. The results suggest that SA-β-gal is a surrogate marker for increased lysosomal activity rather than a specific indicator of senescence.Senescence-associated β-galactosidase (SA-β-gal) is a biomarker for senescent cells, detected at pH 6.0. This study shows that SA-β-gal activity is derived from lysosomal β-galactosidase encoded by the GLB1 gene. In patients with GM1-gangliosidosis, who lack functional lysosomal β-galactosidase, SA-β-gal activity is absent despite replicative senescence. Similarly, reducing GLB1 mRNA levels using RNA interference in normal fibroblasts or HeLa cells led to senescence without SA-β-gal activity. These findings indicate that SA-β-gal activity is not required for senescence but is instead a result of increased lysosomal β-galactosidase levels in senescent cells. The study confirms that lysosomal β-galactosidase is the source of SA-β-gal activity, and that its increased expression in senescent cells contributes to the detection of SA-β-gal at pH 6.0. The results suggest that SA-β-gal is a surrogate marker for increased lysosomal activity rather than a specific indicator of senescence.