Abstract: Ascidians are marine animals with a great ability to synthesize bioactive substances. This study examined the heamolytic potential of ascidians Polyclinum madrasensis and Phallusia nigra. Ascidians P. madrasensis and P. nigra were collected from Tuticorin, Southeast coast of India. The collected ascidians were extracted by methanol at room temperature. The solvents were removed under reduced pressure to give predominantly an aqueous suspension. The protein content of crude P. madrasensis showed 680 μg mL-1 and crude P. nigra showed 543 μg mL-1, respectively. In sodium dodecyl sulfate polyacrylamide gel electrophoresis crude protein of P. madrasensis showed 4 bands 20, 27, 37 and 50 kDa and P. nigra showed 3 bands 20, 37 and 50 kDa. In the hemolytic assay maximum activity 64 Hemolytic Unit was showed in human “B” erythrocytes by P. madrasensis extract and minimum 2 Hemolytic Unit in human ‘O’ erythrocytes by crude extract of P. madrasensis. In 1H-NMR spectrum the spectral signal at δ 8.1 and δ 7.9 indicates the presence of guanidine and thymidine containing alkaloid in P. madrasensis and P. nigra. Thus results proved that the ascidians have remarkable hemolytic compounds and further studies will fulfill for purification and structural elucidation of the compounds.