Updated project metadata. Background: Recent therapeutic advances have greatly improved the eye preservation rate in patients affected by retinoblastoma (RB), however local tumor control remains more difficult in the presence of resistant and/or relapsing retinal or vitreous disease. Thus, the identification of new biomarkers is crucial to design more effective treatment approaches. Recently, technological advances have allowed to safely recover aqueous humor (AH) from patients affected by RB undergoing local treatment. Methods: in order to identify specific proteins associated to active vitreous seeding, we have performed proteomic analysis of AH samples derived from patients affected by RB in different disease stages. Fifty-five AH samples were firstly included in this study. A new widened cohort was then used to validate the proteins identified in the previous discovery set. Results: a total of 808 and 630 proteins were identified in the two analysis, respectively. Five proteins resulted more expressed in the active vitreous seeding group: C19orf57, Girdin, SRFBP, GULP-1 and DCD. These proteins are involved in cytoskeleton regulation, tumor proliferation and growth, invasion and migration of cancer cells. Conclusion: reliable prognostic biomarkers are needed to guide RB management. Proteomic analysis of AH provide a unique opportunity to identify tumor associated biomarkers in patients with RB.