A high-throughput screening campaign using a prefractionated natural product library and an in vitro antimalarial assay identified active fractions derived from the Australian marine sponge Plakortis lita. Bioassay-guided fractionation of the CH2Cl2/CH3OH extract from P. lita resulted in the purification of four novel thiazine-derived alkaloids, thiaplakortones A-D (1-4). The chemical structures of 1-4 were determined following analysis of 1D/2D NMR and MS data. Comparison of the chiro-optical data for 3 and 4 with literature values of related N-methyltryptophan natural products was used to determine the absolute configuration for both thiaplakortones C and D as 11S. Compounds 1-4 displayed significant growth inhibition against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) Plasmodium falciparum (IC50 values <651 nM) and only moderate cytotoxicity against HEK293 cells (IC50 values >3.9 μM). Thiaplakortone A (1) was the most active natural product, with IC50 values of 51 and 6.6 nM against 3D7 and Dd2 lines, respectively. © 2013 American Chemical Society.