Malaria remains a major killer in many parts of the world. Recently, there has been an increase in the role of public-private partnerships inciting academic and industrial scientists to merge their expertise in drug-target validation and in the early stage of drug discovery to identify potential new medicines. There is a need to identify and characterize new molecules showing high efficacy, low toxicity with low propensity to induce resistance in the parasite. In this context, we have studied the structural requirements of the inhibition of PfCDPK1. This is a calcium-dependent protein kinase expressed in Plasmodium falciparum, which has been genetically confirmed as essential for survival. A primary screening assay has been developed. A total of 54000 compounds were tested, yielding two distinct chemical series of nanomolar small molecule inhibitors. The most potent members of each series were further characterized through enzymatic and biophysical analyses. Dissociation rates of the inhibitor-kinase complexes were shown to be key parameters to differentiate both series. Finally, a homology-based model of the kinase core domain has been built which allows rational design of the next generation of inhibitors.