Tyrosyl-DNA phosphodiesterase I (Tdp1) resolves topoisomerase I (Top1)-DNA adducts accumulated from natural DNA damage as well as from the action of certain anticancer drugs. Tdp1 catalyzes the hydrolysis of the phosphodiester bond between the catalytic tyrosine residue of topoisomerase I and the DNA 3'-phosphate. Only a limited number of weak inhibitors have been reported for Tdp1, and there is an unmet need to identify novel chemotypes through screening of chemical libraries. Herein, we present an easily configured, highly miniaturized, and robust Tdp1 assay using the AlphaScreen technology. Uninhibited enzyme reaction is associated with low signal, whereas inhibition leads to a gain of signal, making the present assay format especially attractive for automated large-collection high-throughput screening. We report the identification and initial characterization of four previously unreported inhibitors of Tdp1. Among them, suramin, NF449, and methyl-3,4-dephostatin are phosphotyrosine mimetics that may act as Tdp1 substrate decoys. We also report a novel biochemical assay using the SCAN1 Tdp1 mutant to study the mechanism of action of methyl-3,4-dephostatin.