Phytoplankton biomass and community structure in Southampton Water estuary (on the south coast of the UK) have been investigated. Inter-annual changes in chlorophyll a coupled with changes in total carbon biomass have been analysed in 2004 and 2005 (during the productive period of the estuary). HPLC method has been also used to measure chlorophyll a and other accessory pigments that help, as quantitative biomarkers, to provide information on changing phytoplankton dynamics in such a highly dynamic estuary. Microscopy and chemotaxonomy give a high level of agreement phytoplankton characterization along the estuary; however, some limitations are present in both techniques. HPLC derived chlorophyll a showed a good correlation with the total phytoplankton biomass during the sampling period although it was underestimated in some samples.Fucoxanthin showed a strong correlation with total diatom biomass however high chlorophyll a concentrations during bloom time affected this relationship. Similar finding was obtained for peridinin and Dinoflagellates. Although, Cryptomonas sp. was recorded in some samples, no correlation was detected between its biomass and alloxanthin concentration due to microscopic confusion with small flagellates that were numerically abundant at the same time. Peaks in alloxanthin were, however, coincided with peaks in the biomass of the autotrophic ciliate Mesodinium rubrum. By relating the biomass of specific phytoplankton groups to their corresponding biomarker pigment, the dominance of diatoms (fucoxanthin and chlorophyll c1+c2) in spring and dinoflagellates (peridinin) in summer was established. Dinoflagellates as well as ciliates were found to grow better in the intermediate sites along the estuary. Combining the pigments together to give diagnostic indices shows a very similar pattern to that of Chl a as well as carbon biomass microscopically detected. Diagnostic pigment indices (represented as microplankton, picoplankton and nanoplankton) confirmed the previous finding that phytoplankton species succession in Southampton Water started with diatoms and followed by flagellates/ciliates and then dinoflagellates.