BACKGROUND: Despite significant advances in the treatment of lung cancer (LC), there are no reliable biomarkers to effectively predict therapy response and overall survival (O/S) in non-small cell lung cancer (NSCLC) subtypes. While targeted therapies have improved survival rates in lung adenocarcinoma (LUAD), effective treatment options for lung squamous cell carcinoma (LUSC) are still limited. Recent evidence indicates that exosome-bound WNT5A may significantly contribute to disease progression. Our study assessed the WNT5A protein as a potential biomarker for diagnosing patients and predicting prognosis to assist in therapy selection. METHODS: Primary tumor tissue and serum samples were collected from a cohort of 60 patients with histologically confirmed NSCLC before therapy. Healthy serum donors served as controls. Exosomes were isolated, then exosome number and size were measured, and WNT5A protein levels were identified in tissue and in vesicle-free, vesicle-bound fractions of the serum by ELISA. RESULTS: Extensive statistical analysis (ROC, AUC, Cox, etc.) revealed that elevated WNT5A levels on the serum-exosome surface correlated with distant metastasis, advanced disease stage, and lymph node involvement in LUSC but not in LUAD patients. Moreover, a high WNT5A exosome surface expression was associated with a poor response to therapy and shorter O/S in LUSC patients. Additionally, serum-exosome surface + cargo WNT5A content distinguished LUAD and LUSC subtypes. CONCLUSIONS: WNT5A, particularly its serum exosome-bound form, may serve as a valuable biomarker after further validation for differentiating NSCLC subtypes and predicting disease progression. Importantly, the information can become available from a simple serum sample at the time of diagnosis.