According to recommendations of the international community of phytoplankton functional type algorithm developers, a set of experiments on marine algal cultures was conducted to (1) investigate uncertainties and limits in phytoplankton group discrimination from hyperspectral light absorption properties of assemblages with mixed taxonomic composition, and (2) evaluate the extent to which modifications of the absorption spectral features due to variable light conditions affect the optical discrimination of phytoplankton. Results showed that spectral absorption signatures of multiple species can be extracted from mixed assemblages, even at low relative contributions. Errors in retrieved pigment abundances are, however, influenced by the co-occurrence of species with similar spectral features. Plasticity of absorption spectra due to changes in light conditions weakly affects interspecific differences, with errors <21% for retrievals of pigment concentrations from mixed assemblages.