A proof of concept study is presented with the aim of assessing the feasibility of using aqueous humour surrogate marker for tumour tissue for identifying retinoblastoma RB1 variant. Three children undergoing enucleation for retinoblastoma were recruited from a single centre. Samples of tumour tissue, aqueous humour and blood samples were taken. The authors describe how samples were collected and processed. This study reports that it is possible to use aqueous humour samples as a surrogate marker to obtain cell free DNA from retinoblastoma tumours. The detection in aqueous humour has a relation of tumour burden and it would be recommended that it be obtained prior to commencement of treatment but could also be tested during treatment. Further research is required to validate these findings in larger sample to establish sensitivity and specificity.