Although chemotherapy-induced senescence (CIS) can slow tumor progression by halting the cell cycle, recent studies suggest that some cancer cells may escape this state, resume proliferation, and acquire a more aggressive phenotype. This phenomenon may contribute to chemotherapy resistance in colorectal cancer, highlighting the need to identify which treatments can effectively induce senescence. We previously demonstrated that low doses of SN38 (the active form of irinotecan) and etoposide induce senescence in HCT116 colon cancer cells, accompanied by reprogramming of the Hexosamine Biosynthesis Pathway (HBP) and O-GlcNAcylation. Here, we investigated whether other chemotherapeutic agents also induce senescence in these cells and whether changes in HBP and O-GlcNAcylation are hallmark features of CIS. Our results show that doxorubicin and cisplatin induce senescence, while 5-FU and oxaliplatin do not. Senescence induced by doxorubicin and cisplatin was associated with decreased expression of GFAT (the rate-limiting enzyme of the HBP), OGT, and OGA (the enzymes driving O-GlcNAcylation cycling), along with reduced O-GlcNAcylation levels, consistent with our previous findings. This suggests that HBP reprogramming and O-GlcNAcylation changes are hallmarks of CIS. Furthermore, they highlight the differential ability of chemotherapeutic agents to induce senescence in colorectal cancer cells, which could have implications for optimizing treatment strategies and exploring therapeutic approaches to counteract CIS.