To describe the prospective use of the aqueous humor (AH) as a molecular diagnostic and prognostic liquid biopsy for retinoblastoma (RB).Prospective, observational study wherein an AH liquid biopsy is performed at diagnosis, and longitudinally through therapy for RB patients. Tumor-derived cell free DNA is isolated and sequenced for single nucleotide variant (SNV) analysis of the RB1 gene and to detect somatic copy number alterations (SCNAs). The SCNAs are used to determine tumor fraction (TFx). Specific SCNAs, including 6p gain and focal MycN gain are correlated along with TFx, prospectively, with intraocular tumor relapse, response to therapy and globe salvage.A total of 26 eyes of 21 patients were included with AH taken at diagnosis. Successful ocular salvage was achieved in 19 of 26 (73.1%) eyes. Mutational analysis of 26 AH samples identified 23 pathogenic RB1 variants and 2 focal RB1 deletions; variant allele fraction (VAF) ranged from 30.5% to 100% (median 93.2%). At diagnosis, SCNAs were detectable in 17 of 26 (65.4%) AH samples. Eyes with 6p gain and/or focal MycN gain had significantly greater odds of poor therapeutic outcomes (Odds Ratio [OR]=6.75, 95% CI=1.06-42.84, P =.04). Higher AH TFx was observed in eyes with vitreal progression (TFx=46.0% ± 40.4) than regression (22.0 ± 29.1; difference: -24.0; P = .049).Establishing an AH liquid biopsy for RB is aimed at addressing 1) our inability to biopsy tumor tissue and 2) the lack of molecular biomarkers for intraocular prognosis. Current management decisions for RB are made based solely on clinical features without objective molecular testing. This prognostic study shows great promise for using AH as a companion diagnostic.