Receptor occupancy (RO) assays are designed to quantify the binding of therapeutics to their cell surface targets and are frequently used to generate both pre-clinical and clinical pharmacodynamic biomarker data. Flow cytometry is a commonly used technique to measure receptor occupancy on immune cell populations within fresh blood specimens. Yet, receptor occupancy assays are subject to numerous technical and logistical challenges. To ensure that reliable and high-quality results are generated from receptor occupancy assays, careful assay design and key reagent selection, characterization, and utilization are of critical importance. Antibodies are commonly conjugated with fluorophores and used in receptor occupancy assays for related therapeutics. Bicycles® are a new therapeutic modality - fully synthetic, constrained bicyclic peptides with high affinity and excellent target selectivity. Here, we describe a novel utility for fluorescently-labeled Bicycles in the development of a CD137 (4-1BB) receptor occupancy assay. CD137 is a member of the TNFR superfamily involved in stimulation of several immune cell types, including T cells and NK cells. CD137 is well validated pre-clinically, as agonism with anti-CD137 antibodies is effective in vivo, however, clinical utility to date has been limited by dose dependent hepatotoxicity. We have demonstrated that tumor-targeted immune cell agonists (TICAsTM) comprised of CD137 binding Bicycles coupled to tumor antigen binding Bicycles exert anti tumoral properties with a favorable safety profile. Here, we demonstrate another functionality for the Bicycle platform, as a measurement of CD137 TICA target engagement and receptor occupancy. Using fluorescently-labeled CD137 Bicycles, we measured RO on immune populations in isolated human peripheral blood mononuclear cells (PBMCs), as well as in whole blood. The assay shows high specificity as no Bicycle binding was observed in CD137 negative cell populations. Additionally, RO was shown to be dependent on the presence of CD137-binding Bicycles. Together, this novel approach of measuring receptor occupancy may be used to inform on both the pre-clinical and clinical therapeutic properties of Bicycle TICAs. Citation Format: Drasti N. Kanakia, Punit Upadhyaya, Johanna Lahdenranta, Elizabeth Repash, Eric Haines, Kevin McDonnell, Kristen Hurov, Philip Brandish, Nicholas Keen. Development of a CD137 (4-1BB) receptor occupancy assay using fluorescently labelled Bicycles® [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO076.