Plasmodium falciparum, the deadliest species of malaria parasites, is dependent on glycolysis for the generation of ATP during the pathogenic red blood cell stages. Hexokinase (HK) catalyzes the first step in glycolysis, transferring the γ-phosphoryl group of ATP to glucose to yield glucose-6-phosphate. Here, we describe the validation of a high-throughput assay for screening small molecule collections to identify inhibitors of the P. falciparum HK (PfHK). The assay, which employed an ADP-Glo™ reporter system in a 1536-well plate format, was robust with a signal-to-background of 3.4 ± 1.2, a percent coefficient of variation of 6.8 ± 2.9, and a Z'-factor of 0.75 ± 0.08. Using this assay, we have screened 57,654 molecules from multiple small molecule collections. Confirmed hits were resolved into four clusters based on structural-relatedness. Multiple singleton hits were also identified. The most potent inhibitors had IC50 values as low as ∼1 μM and several were found to have low micromolar EC50 values against asexual intraerythrocytic stage P. falciparum parasites. These molecules additionally demonstrated limited toxicity against a panel of mammalian cells. The identification of PfHK inhibitors with anti-parasitic activity, using this validated screening assay, is encouraging as it justifies additional HTS campaigns with more structurally amenable libraries for the identification of potential leads for future therapeutic development.