Malaria research often requires isolation of individually infected red blood cells (RBCs) or of a homogenous parasite population derived from a single parasite (clone). Traditionally, isolation of individual, parasitized RBCs or parasite cloning is achieved by limiting dilution or micromanipulation. This protocol describes a method for more efficient cloning of the malaria parasite; the method uses a cell sorter to rapidly isolate Plasmodium falciparum-infected RBCs singly. By gating the parameters of forward-angle light scatter and side-angle light scatter in a cell sorter, singly infected RBCs can be isolated and automatically deposited into a 96-well culture plate within 1 min. Including a Percoll purification step; the entire procedure to seed a 96-well plate with singly infected RBCs can take <40 min. This highly efficient single-cell sorting protocol should be useful for cloning of both laboratory parasite populations from genetic manipulation experiments and clinical samples.