Marine phytoplankton fix carbon dioxide during photosynthesis and contribute half of the carbon fixation occurring on Earth. During photosynthesis, small organic molecules are produced within the cell (i.e. endometabolites) and released into the seawater to support heterotrophic organisms in the ocean. Improving our knowledge on phytoplankton endometabolites can bring deeper understanding of phytoplankton metabolism and how it effects the global network of life. The universal problem arises with identifying metabolites from complex mixtures of compounds. Here, we take an approach of making a fraction library where metabolites were separated into fractions by polarity. A common phytoplankton strain Thalassiosira pseudonana (CCMP1335) was used as a representative model for this analysis. Nuclear magnetic resonance (NMR) spectroscopy and a recently developed database matching approach were used for compound annotation. Separating the endometabolome into fractions improves compound identification and supports comprehensive functional analyses that bring us closer to metabolite identification.