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Objective: To evaluate the potential utility of antibody‐drug conjugates targeting trophoblast cell surface antigen‐2 (TROP‐2) in patients with primary penile squamous cell carcinoma (PSCC), patients with recurrence (REC cohort), and patient‐matched distant metastases (MET cohort), and to assess the potential use of TROP‐2 as a predictive non‐invasive biomarker in PSCC. Methods: A cohort comprising a PRIM (n = 37), REC (n = 5) and MET subcohort (n = 7), with MET including lymph node and lung metastases, was analysed using quantitative real‐time PCR, ELISA and immunohistochemical staining with evaluation of H‐score. Results: TROP‐2 mRNA and serum protein levels were significantly increased in primary and recurrent PSCC compared to cancer‐free controls (both P < 0.001). Immunohistochemical analysis revealed that most of the PRIM cohort (n = 34/37, median H‐score 260, interquartile range [IQR] 210–300), as well as all patients in the REC (median [IQR] H‐score 200 [165–290]) and MET cohorts (median [IQR] H‐score 280 [260–300]) exhibited moderate to strong membranous TROP‐2 expression. Additionally, The H‐score (membranous TROP‐2 expression) was positively correlated with TROP‐2 mRNA (ρ = 0.69, P < 0.0001, R2 = 0.70) and protein levels (ρ = 0.86, P < 0.0001, R2 = 0.59), indicating its potential as a non‐invasive biomarker in PSCC. Conclusion: In summary, our results support further studies on TROP‐2 as a diagnostic and therapeutic target in primary, recurrent and metastatic PSCC. [ABSTRACT FROM AUTHOR]