Background Oesophageal adenocarcinoma (OAC) typically presents at an advanced stage with resultant poor survival rates. The 2022 oesophago-gastric (OG) audit suggests only 35.9% of patients diagnosed with OG cancer were treated with curative intent. Raman spectroscopy of serum is a non-invasive and rapid diagnostic technique which has proved promising in cancer diagnostics. A proof-of-concept study of 124 samples undertaken by our team to determine the accuracy of Raman spectral discrimination in detecting oesophageal transformation of Barrett’s to adenocarcinoma revealed sensitivity and specificity of over 95% when using serum to diagnose OAC. Methods Our research group has conducted a further study to validate the aforementioned findings by re-analysing 60 samples from the proof of concept study in a separate laboratory. Chemometric techniques, coupled with principal component analysis, were employed to identify discriminant spectral peaks. Subsequently, a prediction model was developed using a linear discriminant analysis algorithm to accurately classify serum samples from cancer patients. Results Spectral peaks representing methylene deformation at 1450 cm-1 and C-O stretch of ribose at 1012 cm-1 were identified as discriminant spectral peaks in identifying OAC in both analyses, inferring that these results are reproducible. A specificity and sensitivity of 72% and 71% respectively was calculated from chemometric spectral analysis. Conclusions We conclude that Raman spectroscopy has potential applications as a non-invasive screening tool for patients with suspected OAC.