Cellular senescence is a biological process where somatic cells undergo irreversible growth arrest. One key marker of cellular senescence is senescence-associated β-galactosidase (SA-β-gal), an enzyme whose activity can be detected at pH 6 in senescent cells. Traditional methods for SA-β-gal detection often rely on the substrate X-Gal, which requires fixed cells and offers limited sensitivity for quantitative analysis. This study introduces a refined method using the SPiDER β-gal substrate from the Cellular Senescence Detection Kit (Dojindo), designed to improve cell permeability and enable quantification through flow cytometry in both live and fixed cells. We employed this novel protocol to detect SA-β-gal in peripheral blood mononuclear cells (PBMCs) isolated from healthy donors of different ages. The results revealed a progressive increase in SA-β-gal+ CD8+ T cells with advancing age, suggesting that the accumulation of senescent immune cells is a feature of aging. To further validate this method, UVB-treated fibroblasts were analyzed, a well-established model for studying senescence. Overall, this method provides a powerful tool for studying cellular senescence across different cell types and experimental contexts, with broad applications for research on aging, immune function, and cancer biology.