One of the major challenges in imaging bacterial infection using Positron Emission Tomography (PET) is the insufficiency of fluorine-18 labeled probes. Several carbon-11 D-amino acid probes have been developed to mimic bacteria-specific metabolic pathways and have been translated into the clinical realm.1 However, the short half-life of carbon-11 limits its applicability and reach in examining populations of interest. Several attempts to make fluorine-18 probes through direct radiolabeling techniques have failed, and thus a pressing need to fulfill this gap exists. Click chemistry, an indirect alternative tool to tag biomolecules, has been shown to shown to tag and study molecules, including bacteria specific biomarkers such as peptidoglycan.2 Utilizing the cell’s biosynthetic machinery to incorporate essential components to peptidoglycan synthesis such as modified sugars or amino acids, these modified compounds can then selectively ligate to a second probe bearing a visible component.3 By utilizing these well-established bio-orthogonal techniques,4 we developed an optimized, high-throughput azide-alkyne click chemistry assay to indirectly label gram-positive Staphylococcus aureus and gram-negative Escherichia coli with three fluorine-18 PET tracers: [18F]FB-DBCO, [18F]PEG4-DBCO, and [18F]Sulfo-DBCO. Tracer ligation to D-azido-alanine, a modified D-alanine metabolite, via copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) was quantified to determine the efficiency of the fluorine-18 PET tracer and to propose the development of the next generation of PET tracers for infection imaging.