Ascidians, commonly called as Sea Squirts are sedentary tunicates. Phallusia nigra is a simple ascidian belonging to the family Ascidiidae found in plenty throughout the year. The ethanolic extract of Phallusia nigra was subjected to HPTLC and IR spectral analysis to determine the possible bioactive components. In HPTLC studies, gallic acid, ferulic acid, caffeic acid and flavonoids such as rutin, isoquercitrin and quercetin were found to be present. The interpretation of the spectrum showed the presence of aliphatic bromo compounds, phenol or tertiary alcohols, carbonyl compound, carboxylic acids, lipids, proteins, alkanes and aromatic compound.