Cellular senescence is associated with the accumulation of senescent cells characterized by functional alterations, telomere shortening, cell cycle arrest, resistance to apoptosis, and metabolic dysregulation. In recent years, senescence has been extensively investigated not only in the context of aging but also in relation to cancer therapy, as senescence induction in various tumor cell types may differentially influence disease progression. The aim of this study was to comparatively evaluate commonly used chemotherapeutic agents with respect to their ability to induce senescence and their effects on mitochondrial and lysosomal compartments in primary dermal fibroblasts isolated from C57BL/6 mice. Cellular senescence was assessed using both chromogenic and fluorescent assays for β-galactosidase (β-Gal) activity. Mitochondria were labeled with the potential-sensitive dye MitoTracker® Orange, and lysosomes were stained with LysoTracker® Red. Flow cytometry analysis was performed using a BD LSRFortessa cytometer. Our results revealed a significant decrease in mitochondrial membrane potential and an increase in lysosomal fluorescence intensity in cells undergoing chemotherapy-induced senescence. Using an integrative senescence induction index developed in our laboratory, we demonstrated that doxorubicin exerts a more pronounced effect on senescence induction and on mitochondrial and lysosomal compartments compared to cisplatin, bleomycin, and etoposide.