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 that each display restricted amino acid specificities, including PfM1MAA (membrane alanine aminopeptidase), PfM17LAP (leucine aminopeptidase), and PfM18AAP (aspartyl aminopeptidase), which are thought to act in concert to degrade host erythrocyte hemoglobin that the parasite uses as a source of amino acid building blocks for the synthesis of its own proteins. Since there are several small molecule inhibitors of PfM1MAA and PfM17LAP, and very few small molecule inhibitors of PfM18AAP, we set out to identify small molecule inhibitors of PfM18AAP. Biochemical assays employing rPfM18AAP, native PfM18AAP, recombinant Fasciola hepatica cathepsin L1 (rFhCTSL1), rPfM1MAA, rPfM17LAP, and rhM18, and cell-based parasite growth inhibition and cytotoxicity assays were used to identify CID23724194 (from the NIH MLSMR) as a viable starting point for medicinal chemistry optimization. Two rounds of structure-activity relationship studies were performed to generate the probe ML369 (CID 56846691). The probe is the best-in-class small molecule inhibitor of PfM18AAP; however, certain liabilities discussed in detail in the probe report limit its usefulness. When the probe is used as recommended, the probe is “fit-for-purpose” and should be useful for advancing the field.