The aim of this study was to validate ImmunoRatio, a web-based automated image analysis application, by comparing the manual and automated analysis scores for oestrogen receptor α (ERα) in breast carcinomas. Tissue microarrays comprising 200 breast cancer cases prestained for ERα were scanned and scored manually using ImageScope viewing software. Corresponding images were then uploaded and assessed according to the web-based ImmunoRatio programme. There was excellent correlation between manual and ImmunoRatio ERα scores (Spearman correlation=0.872; p≥0.001). The manual and ImmunoRatio ERα scores showed only a moderate agreement (κ=0.421; Weighted kappa=0.874 (CI 0.839 to 0.902)), most probably due to lack of specificity of the algorithm to differentiate between cancer and non-cancer nuclei. Further development to enable differentiation of cancer and non-cancer elements should improve the specificity of the application. Our results support the use of ImmunoRatio software for analysing ERα immunohistochemistry in breast cancer tissues for the purposes of research.