Chronic drug treatment can alter the lipidome of cancer cells, potentially leading to significant biological changes, such as drug resistance or increased tumor aggressiveness. This study examines the lipidome profiles of four human colorectal cancer (CRC) cell lines, comparing treatment-naïve cells with the same cells after chronic exposure to a clinically used combination therapy (FOLFOXIRI: folinic acid, 5-fluorouracil, oxaliplatin, and irinotecan). Lipidomic profiling was obtained with untargeted liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). For data deconvolution and to interpret the multifactorial dataset generated, Analysis of Variance Multiblock Orthogonal Partial Least Squares (AMOPLS) was used. Our results indicate specific shifts in triglycerides (TGs), sphingolipids, and phospholipids in CRC cells resistant to FOLFOXIRI. The overall shift in TGs, phosphatidylcholine, and cholesteryl ester species was notably linked to FOLFOXIRI resistance (-R) in SW620 cells, whereas an increased abundance of phospholipids, mainly hexosylceramide and sphingomyelin, was present in the signatures of HCT116-R, LS174T-R, and DLD1-R cells. These altered lipid species may serve as potential prognostic markers in CRC following chemotherapy. Furthermore, lipid-targeting therapies aimed at reprogramming the lipid profiles of drug-resistant cells could play a crucial role in restoring drug sensitivity and improving patient survival.