Background: Colorectal cancer (CRC) stands as a significant contributor to cancer-related mortality globally. Approximately 90% of cancer mortality is due to distant metastasis. In the context of therapy, response rates can fluctuate significantly with approximately one-third of the patients experiencing relapse following their initial treatment. Owing to its prognostic and therapeutic implications, intratumoral heterogeneity (ITH) presents a considerable challenge in contemporary oncology. In this pre-clinical investigation, we conducted a comprehensive analysis of ITH during the progression of CRC utilizing a single-cell-based spheroid model. Methods: Single cells of the CRC cell lines from a primary colorectal adenocarcinoma (SW480) and a locoregional lymph node metastasis (SW620) were sorted via FACS and cultured into spheroids. Cell culture growth curves were evaluated via microscopy, viability assays were conducted via ATP measurement. A ‘diameter-related metabolic activity’ (DMA) was calculated using the viability and the spheroid diameter. Label-free liquid chromatography mass spectrometry (LC-MS) further assessed ITH in data-independent acquisition mode. Identifying proteins that were most differently expressed between the samples was accomplished through computation and ranking of the fold changes in protein expression data. Chemotherapy response was measured by conducting a viability assay after incubation with 5-Fluorouracil for 72 hours. Results: Single-cell-derived spheroids from both cell lines showed significant differences in morphology, spheroid size, and viability. Subsequently, significant differences in DMA, which could be correlated with aggressive tumor behavior, were observed. Downstream MS-Analysis detected a maximum of 3,954 proteins of which 1,655 were selected for further evaluation. SW620 showed higher proteomic ITH than SW480, which was mediated by distinct pathways. InSW480 cell populations, the implicated pathways comprised tumor suppressor genes, proto-oncogenes, and transcription factors, while SW620 spheroids showed activated pathways of vascularization and the Warburg effect. Furthermore, individual spheroids responded differently to 5-FU chemotherapy showing higher drug resistance in SW620 cells than in SW480 cells. Conclusion: The present study revealed a more pronounced ITH for single-cell-derived spheroids of SW620 regarding spheroid characteristics, proliferation, and proteome activity compared to SW480 spheroids. These findings suggest a prominent involvement of energy metabolism in the metastatic cascade in colorectal cancer models. Further exploration of ITH could provide an important basis for novel biomarker, for personalized tumor therapy and prediction of therapeutic response.