Developing optical detection techniques for discriminating particular phytoplankon species in mixed assemblages has long been a goal of aquatic scientists. Previously, a processing algorithm for phytoplankton absorption spectra was reported that suggested detection of the red tide dinoflagellate Gymnodinium breve was possible. The algorithm evaluated the fourth derivative of the particulate absorption spectrum of an unknown sample and compared it to a standard fourth derivative spectrum for G. breve using a similarity index. We report here the first-time application of this technique to the detection of G. breve in natural, mixed phytoplankton communities. Pigment and spectral absorption data were collected from natural blooms of G. breve in the eastern Gulf of Mexico. This dinoflagellate is the only species of phytoplankton in the Eastern Gulf of Mexico observed to contain the pigment gyroxanthin-diester, and it appears in constant proportion to cellular chlorophyll a (Chl a) in G. breve. The in vitro absorption spectrum of gyroxanthin-diester is nearly identical to other xanthophylls (including diadinoxanthin, lutein, and 199-hexanoyloxyfucoxanthin) and is not singularly responsible for imparting a unique absorption signature. Quantifying gyroxanthin-diester and Chl a allowed us to estimate the fraction of the biomass in mixed populations associated with G. breve. Subsequent regression of the G. breve similarity indexes to the G. breve biomass fractions yielded a significant linear correlation. Finally, the liquid waveguide capillary cell appears to be a promising technology for automating this technique.