Bordetella pertussis causes the highly contagious respiratory disease pertussis, also known as whooping cough. The resurgence of pertussis has been witnessed even in highly vaccinated populations, and macrolide-resistant strains have been isolated. One attractive target for drug development is the pertussis toxin an important type IV secretion system-dependent virulence factor of B. pertussis. The AB5-topology pertussis toxin is composed of a pentameric PtxS2-S5 (1:1:2:1) complex mediating toxin binding to cell surface receptors, and one ADP-ribosyltransferase subunit PtxS1. Once internalized into the host cell, PtxS1 ADP-ribosylates -subunits of heterotrimeric Gi-superfamily members, thereby disrupting G-protein-coupled receptor (GPCR) signaling. Here, we describe protocols to purify mg-levels of truncated but highly active recombinant B. pertussis PtxS1 from E. coli and an in vitro high throughput-compatible assay to quantify NAD+ consumption during PtxS1-catalyzed Gi ADP-ribosylation. The in vitro NAD+ consumption assay was used to screen compounds inhibiting the PtxS1 activity. Two inhibitory compounds (NSC 29193 and NSC 228155) with low micromolar IC50-values were identified that also were potent in an independent in vitro assay monitoring conjugation of ADP-ribose to Gi. Docking and molecular dynamics simulations identified plausible binding poses of NSC 228155 and in particular NSC 29193, most likely owing to the rigidity of the latter ligand, at the NAD+-binding pocket of PtxS1. NSC 228155 inhibited the pertussis AB5 holotoxin-catalyzed ADP-ribosylation of Gi in living human cells in low micromolar concentration. NSC 228155 and NSC 29193 might prove useful as lead compounds in targeted drug development in pertussis.