AIM: To investigate the value of cytokine analysis in aqueous humor (AH) for discriminating vitreoretinal lymphoma (VRL) from uveitis and for evaluating the efficacy of intravitreal methotrexate (MTX) injections. METHODS: This retrospective study was done on 28 VRL patients between 2013 and 2019. AH interleukin (IL)-10, IL-6, IL-8, vascular endothelial growth factor (VEGF), and vascular cell adhesion molecules (VCAM) were measured in 28 VRL patients and 38 uveitis patients. As to the respective examinations for distinguishing VRL from uveitis, the diagnostic accuracy was evaluated by receiver operating characteristic (ROC) curve analysis. The response to treatment was monitored by observing changes in best-corrected visual acuity (BCVA), ocular manifestation, and AH cytokine levels in 21 patients with VRL who had undergo multiple intravitreal injections of MTX. RESULTS: Compared with uveitis patients, VRL patients had higher IL-10 level (P<0.001) and IL-10/IL-6 ratio (P<0.001), whereas patients with uveitis had significantly higher IL-6 level than those with VRL (P=0.003). An ROC analysis was used to identify the diagnostic threshold values for VRL, and it was found that optimal sensitivity and specificity improved to 94.1% and 100%, respectively, for IL-10/IL-6>1.55 and 88.2% and 81.1%, respectively, for IL-10>76.7 pg/mL. In 21 patients who had undergo repeated injections, improvements in BCVA, clinical remission of VRL and continuous decrease in cytokine levels over time were observed. In those patients, the BCVA correlated with the aqueous levels of IL-10 and IL-6 during the course of disease treatment. CONCLUSION: The combination of the aqueous cytokine profiles can be instrumental for conventional diagnostic methods and for progression monitoring and treatment response.