Malaria is one of the most prevalent human parasitic diseases and is a global health issue accounting for >600,000 deaths annually. For survival, the Plasmodium falciparum (Pf) malaria parasite requires the action of a number of metallo-aminopeptidases, including PfM1MAA (membrane alanine aminopeptidase), PfM17LAP (leucine aminopeptidase), and PfM18AAP (aspartyl aminopeptidase). Each enzyme displays restricted amino acid specificity, and they are thought to act in concert to degrade proteins (i.e., host erythrocyte hemoglobin) that the parasite uses to generate a pool of amino acids which are employed as building blocks for the synthesis of its own proteins. Since there were very few small-molecule inhibitors of PfM17LAP and no selective inhibitors relative to PfM1AAP (or PfM18AAP), we set out to identify new potent and selective small-molecule inhibitors of this enzyme. Biochemical assays employing enzymatically active recombinant PfM17LAP (rPfM17LAP), as well as recombinant Fasciola hepatica cathepsin L1 (rFhCTSL1), rPfM1MAA, rPfM18AAP, and human M17LAP (rhuM17), and cell-based parasite growth inhibition and cytotoxicity assays were used to identify CID 2466 (from the NIH MLSMR) as a viable starting point for SAR analysis. Three rounds of structure-activity relationship studies were performed to generate a panel of probe candidate compounds. Ultimately, the compound hit, CID 2466, also known as bufexamac, was nominated as the probe ML392. When the probe and analogues are used as recommended, they are “fit-for-purpose” and should be useful for advancing the search for new antimalarial drugs directed at PfM17LAP.