Prostate-specific membrane antigen (PSMA) is a promising theranostic target. Different PSMA-targeting small molecule ligands have been FDA-approved or are in development, yet their biological fate at the single-cell level is often unknown. An improved understanding of the cellular distribution of these probes will confer insights into their microdosimetry and guide next-generation theranostic probe development. To enable detailed single-cell pharmacokinetics, it is desirable to have fluorescence affinity ligands that preserve the properties of the native agent. Building upon the structure of the FDA-approved PSMA-617, we synthesized a panel of fluorescent analogs and evaluated their in vitro and in vivo properties. We described a facile solid-phase-based synthesis and optimized the synthesis of the crucial urea pharmacophore. We identified two compounds, PSMA-Lys-DOTA-Cy680 (3) and PSMA-Lys-DOTA-AF647 (4), with similar PSMA binding affinities compared to the parent compound and robust optical imaging properties. Tissue and cellular biodistribution data from imaging can populate microdosimetric and systemic modeling to provide potential insights into future radiopharmaceutical therapy design.