Significance Malaria remains a major global health threat. In the face of increasing resistance to available chemotherapeutics, new antimalarial drugs with new modes of action are urgently needed. The causative agent of malaria is a single-celled parasite that invades and replicates within red blood cells. Escape from the red cell, a process called egress, involves a proteolytic pathway triggered by an essential parasite subtilisin-like serine protease called SUB1. Here, we describe the development and rational optimization of a potent, membrane-permeable substrate-based boronic acid compounds that block egress and parasite proliferation by direct inhibition of SUB1 activity. The compounds could form the basis of a new type of antimalarial medicine that would both protect against infection and treat disease.