Fluorescence-guided surgery is an emerging field with potential to improve surgical outcomes. We are currently investigating tumour targeting fluorescent probes known as quenched activity-based probes (qABP) which operate by covalently bonding to tumour-expressed proteases known as cathepsins. Fluorescence is inhibited up until the qABP bonds to cathepsins, which releases an inhibitory quencher, resulting in emission of the fluorescent signal. This improves the contrast between normal tissue and tumours. We sought to test the qABP’s (BMV109 and VGT309) in oesophageal, junctional and gastric cancer. Cathepsin activity was screened in patient-derived cell lines from the normal oesophagus, Barrett’s metaplasia, oesophageal adenocarcinoma (OAC), squamous cell carcinoma (SCC) and gastric adenocarcinoma (GAC) with BMV109 and VGT309. We additionally screened biopsies collected from patients with oesophageal, junctional and gastric cancers. Cell line and tissue samples were analysed using in-gel fluorescence (Amersham Typhoon) and Western blot. In-vivo, cell line xenografts were established in NSG mice using models that demonstrated cathepsin activity in-vitro. Tumour-bearing mice were then injected with VGT-309 and then imaged at specific timepoints using the IVIS spectrum imager. Cathepsin activity was found to be active in all oesophago-gastric cell lines. Selectivity was proven by eliminating activity using an cathepsin inhibitor. Matched biopsies were collected from 50 patients prior to commencement of neoadjuvant therapy. OAC and GAC samples had significantly higher cathepsin activity than normal oesophageal and gastric samples (p = <0.001). In-vivo, GAC xenografts were able to be identified in NSG mice as early as 12 hours post injection of VGT-309 and could differentiated from the surrounding normal tissue. Bio-distribution analysis demonstrated that VGT-309 accumulated in liver, kidney and lung, but less so in the murine oesophagus and stomach. Our initial work demonstrates that qABP’s are active towards cancers of the oesophagus, junction and stomach and can differentiate tumour from normal surrounding tissue. Translating qABP’s into the operating room has the potential to detect early cancers in Barrett’s metaplasia, reduce positive margin rates and identify lymph node metastasis. Our research group continues to investigate qABP’s to determine their sensitivity to detect lymph node metastasis and identify any effects of neo-adjuvant treatment on probe activity.