Surfactin-type phosphopantetheinyl transferases (Sfp-PPTases) are responsible for modifying type I polyketide and non-ribosomal peptide synthases of prokaryotes and have been implicated in the activation of a variety of pathogen-associated virulence factors. As such, inhibitors of this enzyme class represent enticing leads for antibiotic development and can serve as tools in studies of bacterial metabolism. Currently, no small molecule inhibitors of Sfp-PPTase are known, highlighting the need for efficient methods for PPTase inhibitor identification and development. Herein, we present the design and implementation of a robust and miniaturized high-throughput kinetic assay for inhibitors of Sfp-PPTase using the substrate combination of rhodamine-labeled coenzyme A and Black Hole Quencher-2 labeled consensus acceptor peptide YbbR. Upon PPTase-catalyzed transfer of the rhodamine-labeled phosphopantetheinyl arm onto the acceptor peptide, the fluorescent donor and quencher are covalently joined and the fluorescence signal is reduced. This assay was miniaturized to a low 4 microL volume in 1536-well format and was used to screen the library of pharmacologically active compounds (LOPAC(1280)). Top inhibitors identified by the screen were further characterized in secondary assays, including protein phosphopantetheinylation detected by gel electrophoresis. The present assay enables the screening of large compound libraries against Sfp-PPTase in a robust and automated fashion and is applicable to designing assays for related transferase enzymes.