Accumulation of senescent cells with age leads to tissue dysfunction and related diseases. The detection of these cells in vivo remains a challenge in aging research. This study describes the development of a fluorogenic probe (sulfonic-Cy7Gal) based on a galactose derivative, which serves as a substrate for β-galactosidase. The probe is conjugated to a Cy7 fluorophore modified with sulfonic groups to enhance its ability to diffuse. When administered to mice, β-galactosidase cleaves the O-glycosidic bond, releasing the fluorophore, which is excreted by the kidneys and can be measured in urine. The intensity of the recovered fluorophore reliably reflects an experimentally controlled load of cellular senescence and correlates with age-associated anxiety during aging and senolytic treatment. Interestingly, the findings indicate that the effects of senolytic treatments are temporary if the treatment is discontinued. This strategy may serve as a basis for developing fluorogenic platforms for easy longitudinal monitoring of enzymatic activities in biofluids. The design and synthesis of the probe are detailed, along with its validation in cell cultures and in vivo studies using mouse models of aging and senolytic intervention. The probe demonstrates reliable monitoring of β-galactosidase activity and provides insights into the transient nature of senolytic treatments.Accumulation of senescent cells with age leads to tissue dysfunction and related diseases. The detection of these cells in vivo remains a challenge in aging research. This study describes the development of a fluorogenic probe (sulfonic-Cy7Gal) based on a galactose derivative, which serves as a substrate for β-galactosidase. The probe is conjugated to a Cy7 fluorophore modified with sulfonic groups to enhance its ability to diffuse. When administered to mice, β-galactosidase cleaves the O-glycosidic bond, releasing the fluorophore, which is excreted by the kidneys and can be measured in urine. The intensity of the recovered fluorophore reliably reflects an experimentally controlled load of cellular senescence and correlates with age-associated anxiety during aging and senolytic treatment. Interestingly, the findings indicate that the effects of senolytic treatments are temporary if the treatment is discontinued. This strategy may serve as a basis for developing fluorogenic platforms for easy longitudinal monitoring of enzymatic activities in biofluids. The design and synthesis of the probe are detailed, along with its validation in cell cultures and in vivo studies using mouse models of aging and senolytic intervention. The probe demonstrates reliable monitoring of β-galactosidase activity and provides insights into the transient nature of senolytic treatments.