High-throughput screening is utilized by pharmaceutical researchers and, increasingly, academic investigators to identify agents that act upon enzymes, receptors, and cellular processes. Screening hits include molecules that specifically bind the target and a greater number of non-specific compounds. It is necessary to 'triage' these hits to identify the subset worthy of further exploration. As part of our antibacterial drug discovery effort, we applied a suite of biochemical and biophysical tools to accelerate the triage process. We describe application of these tools to a series of 9-oxo-4,9-dihydropyrazolo[5,1-b]quinazoline-2-carboxylic acids (PQ) hits from a screen of Escherichia coli phosphopantetheine adenylyltransferase (PPAT). Initial confirmation of specific binding to phosphopantetheine adenylyltransferase was obtained using biochemical and biophysical tools, including a novel orthogonal assay, isothermal titration calorimetry, and saturation transfer difference NMR. To identify the phosphopantetheine adenylyltransferase sub-site bound by these inhibitors, two techniques were utilized: steady-state enzyme kinetics and a novel (19)F NMR method in which fluorine-containing fragments that bind the ATP and/or phosphopantetheine sites serve as competitive reporter probes. These data are consistent with PQs binding the ATP sub-site. In addition to identification of a series of PPAT inhibitors, the described hit triage process is broadly applicable to other enzyme targets in which milligram quantities of purified target protein are available.