A method for a qualitative analysis of sea urchin diet is based on the characterization of the photosynthetic pigment indices of the major algal groups in the sea urchin gut. The pigments were separated by reverse-phase high performance liquid chromatography (RP-HPLC). This study demonstrated that HPLC is a better method to estimate chlorophyll-a in the gut contents than the conventional spectrophotometric methods which overestimate the amounts by including chlorophyll-a breakdown products. Three sea urchins species, Paracentrotus lividus, Psammechinus miliaris and Sphaerechinus granularis, settled on the loose-lying coralline algae (maerl) in the Bay of Brest (France), were used in this study. The algal pigments identified within the gut contents included chlorophylls-a, -b, -c, fucoxanthin, lutein, βε-carotene and ββ-carotene. The presence of chlorophylls and carotenoid biomarkers was used to characterize the three algal groups: Rhodophyceae, Chlorophyceae, Phaeophyceae in estimating sea urchin diet. The pigment analysis reported here demonstrated that the three species of sea urchins investigated mainly consumed Rhodophyceae which dominate the epibenthic flora in the study area.