Background: In ulcerative colitis, the complexity of mucosal cytokine secretion profiles and how they correlate with endoscopic and clinical scores is still unclear. Methods: In this study, we collected fresh biopsies from UC patients to investigate which cytokines are produced in ex vivo culture conditions, a platform increasingly used for testing of novel drugs. Then, we correlated cytokine production with several scoring indices commonly used to assess the severity of the disease. Results: Increased levels of IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, TNFα and IFNɣ were produced by biopsies of UC patients compared to non-IBD controls. Our results show a better correlation of cytokine levels with Mayo Endoscopic Subscore (MES) and Mayo score, than the more complex Ulcerative Colitis Endoscopic Index of Severity (UCEIS). Out of 10 measured cytokines, eight correlated with MES, six with Mayo score and only three with UCEIS, due to the partial increase in cytokine secretion observed in donors with UCEIS = 7–8. When we analysed individual subscores within the UCEIS, Vascular Network subscore showed a correlation similar to MES (7/10 cytokines), while Bleeding as well as Erosions and Ulcers subscores correlated with only 3/10 cytokines, similarly to the total UCEIS. Conclusions: Our findings suggest that choosing biopsies from donors with MES = 2–3 and UCEIS = 2–6 from areas with no bleeding and no superficial and/or deep ulcers could enable a deeper insight into the cytokine profile of the inflamed tissue and represent a better tool for studying potential therapeutic targets and evaluation of novel therapies.