Malaria is a devastating infectious disease, which causes over 400,000 deaths per annum and impacts the lives of nearly half the world's population. The causative agent, a protozoan parasite, replicates within red blood cells (RBCs), eventually destroying the cells in a lytic process called egress to release a new generation of parasites. These invade fresh RBCs to repeat the cycle. Egress is regulated by an essential parasite subtilisin-like serine protease called SUB1. Here, we describe the development and optimization of substrate-based peptidic boronic acids that inhibit Plasmodium falciparum SUB1 with low nanomolar potency. Structural optimization generated membranepermeable, slow off-rate inhibitors that prevent P. falciparum egress through direct inhibition of SUB1 activity and block parasite replication in vitro at submicromolar concentrations. Our results validate SUB1 as a potential target for a new class of antimalarial drugs designed to prevent parasite replication and disease progression.