Background: Senolytic therapy has garnered significant attention for its potential in combating cancer. This study aimed to investigate the effects of three combinations of chemotherapeutic drugs with well-known senolytic agents (cisplatin+metformin, irinotecan+quercetin, oxaliplatin+caffeine) in K-RAS mutant (HCT-116 and CT26) and wild-type K-RAS-expressing (SW48) colorectal cancer (CRC) cells to assess the impact of K-RAS mutations on the efficacy of these senolytic therapies. Methods: The optimal combination treatment schedule was determined for each chemotherapeutic drug and adjuvant to maximize their senotherapeutic efficacy. Senescence was evaluated through SA-β-gal staining, p16 and p21 expression, and nuclear morphology. Cell viability and death were assessed using the MTT assay, fluorescent staining methods, annexin V/PI flow cytometry analysis, and RT-qPCR analyses for Bcl-2, Bax, and beclin1. Results: Cisplatin (5 µM), irinotecan (2.5 µM), and oxaliplatin (2 µM) alone induced multiple senescence hallmarks, including increased SA-β-gal activity, p16 and p21 upregulation, and multinucleation, in all tested cells. The post-treatment of cisplatin-treated cells with 5 mM metformin (cisplatin+metformin), the combined treatment of irinotecan and 25 µM quercetin (irinotecan+quercetin), and the post-treatment of oxaliplatin-treated cells with 2 mM caffeine (oxaliplatin+caffeine) resulted in decreased numbers of SA-β-gal+ cells, reduced cell viability, and lower p16 mRNA expression, alongside increased annexin V-positive cells, Bax/Bcl-2 ratio, and beclin-1 levels compared to drug-alone treatments. Acridine orange (AO)/ethidium bromide and AO staining confirmed the induction of apoptosis and autophagy in CRC cells for all schedules. The senotherapeutic efficacy was ranked as follows: cisplatin+metformin ≥ irinotecan+quercetin > oxaliplatin+caffeine.

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