In conclusion, our study strongly indicates that a diagnostic system based on combination of PESI-MS and a machine learning-based algorithm can be used to assist the rapid detection of tumour areas with activated TGF-β signalling in HNSCC tumour tissue without conventional immunohistological examination, and has the potential to be a feasible method to diagnose intratumoural phenotypic heterogeneity of TGF-β signalling at the bedside. This evidence concerns the gene TGFB1 and head and neck squamous cell carcinoma.