Sambiloto (Andrographis paniculata) has been used to treat malaria. In vitro research using parasite or through heme polymerization inhibition using andrografolide and in vivo using infected animal test have been done widely, however, heme polymerization inhibition from extracts with different polarity levels has not been studied yet. The aims of this study were to investigate the heme polymerization inhibition activity and toxicity of sambiloto leaf extracts. Sambiloto leaf extracted with gradually maceration using n-hexane, ethyl acetate, and 70% ethanol respectively. Heme polymerization inhibition activity was used as in vitro antimalarial test. Brine shrimp lethality test (BSLT) was used to determine toxicity of the extracts. Phytochemically screening was done for all extracts qualitatively. The results of this study were n-hexane, ethyl acetate, and 70% ethanol extracts had heme polymerization inhibition activities with IC50 values at 2,196.57; 1,235.54; and 1,157.24 μg/mL respectively. N-hexane, ethyl acetate, and 70% ethanol have LC50 values at 1,155.79; 1,133.89; and 5,229.15 μg/mL respectively. 70% ethanol extract contains alkaloid, flavonoid, steroid/triterpenoid, saponin, and tannin. N-hexane and ethyl acetate extracts has only contains alkaloid and flavonoid. 70% ethanol, ethyl acetate, and n-hexane extracts of sambiloto leaf have ability to inhibit heme polymerization and also non toxic to Artemia salina larvae.  Abstrak Sambiloto (Andrographis paniculata) telah lama digunakan untuk mengobati malaria. Penelitian in vitro dengan parasit maupun melalui penghambatan polimerisasi hem dengan senyawa andrografolida dan in vivo dengan hewan uji terinfeksi telah dilakukan, namun demikian belum ada penelitian tentang penghambatan polimerisasi hem ekstrak dengan tingkat kepolaran yang berbeda. Penelitian ini bertujuan untuk mengetahui aktivitas penghambatan polimerisasi hem dan toksisitas ekstrak daun sambiloto. Daun sambiloto diekstraksi secara maserasi bertingkat berturut-turut menggunakan pelarut n-heksan, etil asetat, dan etanol 70%. Uji antimalaria secara in vitro menggunakan metode penghambatan polimerisasi hem. Uji toksisitas menggunakan metode uji kematian larva Artemia salina (BSLT). Skrining fitokimia dilakukan secara kualitatif untuk seluruh ekstrak. Hasil penelitian menunjukkan bahwa ekstrak n-heksan, etil asetat, dan etanol 70% memiliki aktivitas penghambatan polimerisasi hem dengan nilai IC50 masing-masing sebesar 2.196,57; 1.235,54; dan 1.157,24 μg/mL. Ekstrak n-heksan, etil asetat, dan etanol 70% masing-masing memiliki nilai LC50 sebesar 1.155,79; 1.133,89; dan 5.229,15 μg/mL. Ekstrak etanol 70% mengandung alkaloid, flavonoid, steroid/triterpenoid, saponin, dan tannin. Ekstrak n-heksan dan etil asetat hanya mengandung alkaloid dan steroid/triterpenoid. Ekstrak etanol 70%, etil asetat, dan n-heksan daun sambiloto memiliki kemampuan dalam menghambat polimerisasi hem dan tidak toksik terhadap larva Artemia salina.