Marine algae are rich sources of natural bioactive compounds. Out of the three important macoalgae, viz. red, brown and green, brown macroalgae are most commonly targeted for screening of antioxidant compounds. These compounds reported to play important roles against diseases through protection of cells from oxidative damages. The aim of this study is to identify antioxidant compounds of marine algal origin and characterize those using analytical techniques such as HPLC, TLC and Mass spectrometry. In this study, biomass of Stoechospermum polypodioides, a brown macroalga was subjected to sequential solvent extraction followed by mass spectrometric characterization. LC-MS profile of the chloroform fraction shows a peak at 659.53, m/z indicating the presence of fucoxanthin pigment. The fucoxanthin fraction was further isolated and purified using a reversed-phase C-18 (RP-18) column coupled to an HPLC. Additionally, MS/MS analyses together with UV-Visible spectra confirmed the presence of fucoxanthin. The antioxidant activity of fucoxanthin was determined by employing DPPH assay. The algal extracts (in methyl ethyl ketone, petroleum ether and methanol) on ESI MS/MS analysis further revealed the presence of neoxanthin, canthaxanthin, pheophorbide, pheophytin a, and lycopene pigments with potential antioxidant properties. The fragmentation behavior of these molecules in positive ionization mode ESI MS/MS was further examined.