Flow cytometry data allow the direct calculation of picoplancton cell abundance and relative fluorescence, and an indirect estimation of their cell size. The measure of extracted chlorophyll in the <3 µm size fraction give a good estimation of the picoplankton biomass and its contribution to total phytoplankton biomass. The red fluorescence (RF) measured by flow cytometry is a proxy for chlorophyll as evidenced by a simple correlation between chlorophyll <3 µm and total RF. Therefore, the contributions of the 3 picoplankton groups (Prochlorococcus, Synechococcus and picoeukaryotes) to the phytoplankton biomass can be assessed from their RF and their specific chlorophyll content per unit of red fluorescence (F). If the number of replicates is large enough (>4), F values can be calculated by regression using measured values of chlorophyll and picoplankton groups RF. An application of this method was made on atoll lagoon data and its validity is discussed.