BACKGROUND: The Bicycle® toxin conjugate (BTC) zelenectide pevedotin, formerly known as BT8009, is a novel bicyclic peptide targeting the Nectin-4 tumor antigen conjugated to the cytotoxin monomethyl auristatin E (MMAE) via a valine-citrulline cleavable linker. Zelenectide pevedotin is currently being investigated in a Phase 1/2 (Duravelo-1, NCT04561362) clinical trial to determine safety and efficacy in patients with tumors associated with Nectin-4 expression. A simple regulated bioanalytical assay was developed to quantify intact zelenectide pevedotin in patient plasma samples. METHODOLOGY: Quantitation of the intact zelenectide pevedotin and its analog internal standard BCY6063 encompassed a routine protein precipitation procedure followed by reverse phase chromatographic separation paired with tandem mass spectrometric detection. RESULTS: Intact zelenectide pevedotin was quantified over the assay range of 5-2500 ng/mL using 50 µL of human plasma. The method was validated according to local standard operating procedures and has met all US Food and Drug Administration and European Medicines Agency regulatory guidance criteria. CONCLUSION: A bioanalytical method for measuring zelenectide pevedotin in plasma samples was developed and successfully applied in evaluating over 3000 samples from patients in a Phase 1/2 clinical study of zelenectide pevedotin in patients with advanced solid tumors.