Protein kinases (PKs) present in Plasmodium falciparum catalyze phosphorylation reaction to control growth and differentiation of the parasite throughout the life cycle. Protein kinase inhibitors are found to kill the parasite but their cellular target enzymes are not known. Protein kinase inhibitors are evaluated in an in sillico docking studies using plasmodium falciparum RIO-2 kinase (right open reading frame-2 protein kinase) as target enzyme. Most of the protein kinase inhibitors showed appropriate docking within the ATP binding domain of the PfRIO-2 kinase. The initial docking experiments were further validated by a substrate competition experiment to validate the preliminary screening results and test the potentials of these inhibitors under in vivo conditions. Docking and substrate competition study identifies wortmannin, enzastaurin, indirubin-3'-monoxime, apigenin, kaempferol and 8-hydroxy-4-methyl-9-nitro-2H-benzo[g]chromen-2-one as lead inhibitors against native/active form of the PfRIO-2 kinase. The top protein kinase inhibitors bind into the ATP binding site with a similar conformation as ATP. The docking result is in good agreement with the antimalarial schizonticidal IC50 (μg/ml) of an inhibitor and gives a correlation factor (R2) of 0.82 whereas top hit antimalarial inhibitors gives a correlation factor (R2) of 0.99. In summary, our work highlights the importance of PfRIO-2 kinase as a target behind the antimalarial action of protein kinase inhibitors and might help to design a new set of antimalarial remedies.