Cysteine proteases have been implicated in a variety of processes essential for the survival and progression of the malarial parasite Plasmodium falciparum. Here, we synthesized a cysteine protease inhibitor that contains the electrophilic aziridine-2,3-dicarboxylic acid as the reactive agent and biotin as a targeting label. Diethyl ester and dibenzyl ester derivatives of the inhibitor were active against cathepsin L and the plasmodial protease falcipain 2, but only the latter displayed potent antiplasmodial activity against viable parasites. The morphological changes observed during the intraerythrocytic life stages of Plasmodium suggest that degradation of hemoglobin of the host cell is seriously affected, eventually leading to growth arrest and cell death of the parasites. After incubation of infected erythrocytes with the compound plasmodial proteins were captured, with the biotinyl group of the inhibitor serving as an affinity tag. Among these the cysteine proteases falcipain 2 and falcipain 3 were identified as potential target proteins of the compound as evidenced by tandem mass spectrometry. Apparently, the compound gets access to intracellular compartments and therein targets plasmodial cysteine proteases. Accordingly, the reagent described here appears to be a valuable template to develop cell-permeable, non-radioactive reagents that selectively target enzymes involved in pathogenicity of the parasite.